Apparatus and method for analysis of molecules

ABSTRACT

The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Utility patentapplication Ser. No. 10/944,106, filed Sep. 17, 2004, pending, which ishereby incorporated herein by reference in its entirety for allpurposes. This application also claims priority to U.S. ProvisionalApplication Nos. 60/649,009 and 60/651,846 filed on Jan. 31, 2005 andFebruary 9, 2005, respectively, both of which are incorporated herein byreference for all purposes.

BACKGROUND OF THE INVENTION

Confinement of illumination and signal detection has long beenrecognized as an important tool in molecular diagnostics since theapplication of Fluorescence Correlation Spectroscopy (FCS). FCS involvesillumination of a sample volume containing fluorophore-labeledmolecules, and detection of fluctuations in fluorescence signal producedby the molecules as they diffuse into and out of an effectiveobservation volume. The fluorescence intensity fluctuations can best beanalyzed if the volume under observation contains only a small number offluorescent molecules, and if the background signal is low. This can beaccomplished by the combination of a drastically limited detectionvolume and a low sample concentration. The detection volume oftraditional FCS is approximately 0.5 femtoliters (or 0.5×10⁻¹⁵ liters),and is achieved through the use of a high numerical aperture microscopeobjective lens to tightly focus a laser beam. In this detection volume,single molecules can be observed in solutions at concentrations of up toapproximately one nanomolar. This concentration range is unacceptablylow for most biochemical reactions, which have reaction constants thatare typically in or above the micromolar range. At lower concentrations,these reactions either do not proceed acceptably fast, or behave in aqualitatively different fashion than is useful in most analyses. Toobserve single molecules at higher, more relevant concentrations, theobservation volume would typically need to be reduced to far smallerdimensions.

In recent years, the advancement in nanofabrication technology enabledthe production of nanoscale devices that are integrated with electrical,optical, chemical and/or mechanical elements.

However, there still remains a considerable need for chemical andbiological analyses that are faster, cheaper and of greater accuracy, toprovide for the ability to observe single molecule reactions underconditions that are more biologically or diagnostically relevant. Therealso exists a need for small, mass produced, and disposable devices thatcan aid in these goals by providing optical confinements that areamenable to single-molecule analysis at a higher concentration. Thepresent invention satisfies these needs and provides related advantagesas well.

SUMMARY OF THE INVENTION

A principal aspect of the present invention is the design of opticaldevices and methods for characterizing molecules and/or monitoringchemical reactions. The devices and methods of the present invention areparticularly suited for single-molecule analysis.

Accordingly, the present invention provides an array of opticalconfinements having a surface density exceeding 4×10⁴ confinements permm², preferably exceeding 10⁵ confinements per mm². In one aspect, theindividual confinement in the array provide an effective observationvolume that is less than one nanoliter (10×⁻⁹ liter), less than onepicoliter, or less than one femtoliter, preferably on the order ofzeptoliter. In other aspects, each of the individual confinementprovides an effective observation volume that is less than 1000zeptoliters, 100 zeptoliters, 80 zeptoliters, or less than 50zeptoliters, or even less than 10 zeptoliters.

In other aspects, each of the individual confinement yields an effectiveobservation volume that permits resolution of individual moleculespresent at a concentration that is higher than one nanomolar, or higherthan 100 nanomolar, or on the order of micromolar range. In certainpreferred aspects, each of the individual confinement yields aneffective observation volume that permits resolution of individualmolecules present at a physiologically relevant concentration, e.g., ata concentration higher than about 1 micromolar, or higher than 50micromolar range or even higher than 100 micromolar. The array maycomprise a zero-mode waveguide or other nanoscale optical structure. Thearray of optical confinements may further comprise another array ofconfinements that does not yield the above-described effectiveobservation volume or does not permit resolution of individualmolecules. For example, the array of optical confinements may be coupledwith or integrated into a microtiter plate, where a separate array ofoptical confinements may be disposed within each of several differentwells on a multiwell reaction plate. The array of optical confinementmay comprise at least about 2X10⁵ optical confinement, or at least about10⁶, or at least about 10⁷ optical confinements.

In another embodiment, the present invention provides a method ofcreating a plurality of optical confinements having the aforementionedcharacteristics. The method involves the steps of (a) providing asubstrate; and (b) forming an array of optical confinements having asurface density exceeding 4×10⁴ confinements per mm², wherein theindividual confinement comprises a zero-mode waveguide comprising: acladding surrounding a core, wherein said cladding is configured topreclude propagation of electromagnetic energy of a wavelength longerthan a cutoff wavelength longitudinally through the core of thezero-mode waveguide; and (c) illuminating the array with anelectromagnetic radiation of a frequency less than the cutoff frequency,thereby creating the plurality of optical confinements.

In another embodiment, the present invention provides a method ofcreating an optical observation volume that permits resolution ofindividual molecules. The method involves providing a zero-modewaveguide that comprises a cladding surrounding a core, wherein saidcladding is configured to preclude propagation of electromagnetic energyof a frequency less than a cutoff frequency longitudinally through thecore of the zero-mode waveguide, wherein upon illuminating the zero-modewaveguide with an electromagnetic radiation of a frequency less than thecutoff frequency, the zero-mode waveguide yields an effectiveobservation volume that permits resolution of individual molecules. Incertain aspects, the effective observation volume is less than onenanoliter (10⁻⁹ liter), less than one picoliter, or less than onefemtoliter, preferably on the order of zeptoliters. Using the zero-modewaveguide of the present invention, one typically can obtain aneffective observation volume that is less than 100 zeptoliter (100×10⁻²¹liters) or less than 50 zeptoliters, or even less than 10 zeptoliters.In other aspects, the method yields an effective observation volume thatpermits resolution of individual molecules present at a concentrationthat is higher than one nanomolar, more often higher than 100 nanomolar,and preferably on the order of micromolar range. In preferredembodiments, individual molecules present at a concentration higher thanabout 5 micromolar, or higher than 7.5 micromolar, or even higher than50 micromolar range, can be resolved by the method of the presentinvention.

The present invention also provides a method of detecting interactionsamong a plurality of molecules. The method comprises the steps of (a)placing the plurality of molecules in close proximity to an array ofzero-mode waveguides, wherein individual waveguides in the array areseparated by a distance sufficient to yield detectable intensities ofdiffractive scattering at multiple diffracted orders upon illuminatingthe array with an incident wavelength; (b) illuminating the array ofzero-mode waveguides with an incident wavelength; and (c) detecting achange in the intensities of diffractive scattering of the incidentwavelength at the multiple diffracted orders, thereby detecting theinteractions among a plurality of molecules.

The present invention also provides a method of reducing diffractivescattering upon illuminating an array of optical confinements with anincident wavelength, wherein the array comprises at least a firstoptical confinement and a second optical confinement, said methodcomprising: forming the array of optical confinements wherein theoptical confinement is separated from the second optical confinement bya distance such that upon illumination with the incident wavelength,intensity of diffractive scattering resulting from the first opticalconfinement at a given angle is less than that if the first opticalconfinement were illuminated with the same incident wavelength in theabsence of the optical confinement. In preferred aspects, theaforementioned optical confinements are zero mode waveguides.

The present invention also includes a method of detecting a biologicalanalyte using an array of optical confinements having a density on asubstrate exceeding 4×10⁴ confinements per mm² or any other densitydescribed herein or equivalents thereof. The method typically involvesilluminating at least one optical confinement within the array that issuspected to contain the analyte with an incident light beam. Theinvention also provides a method of using of an array of opticalconfinements having a density on a substrate exceeding 4×10⁴confinements per mm² any other density described herein or equivalentsthereof for performing multiple chemical reactions. The method comprisesthe steps of placing the plurality of reaction samples comprisinglabeled reactants into the optical confinements in the array, wherein aseparate reaction sample is placed into a different confinement in thearray; subjecting the array to conditions suitable for formation ofproducts of the chemical reactions; and detecting the formation of theproducts with said optical system.

In addition, the invention provides a method of sequencing a pluralityof target nucleic acid molecules. The method typically involves (a)providing an array of optical confinements having a density on asubstrate exceeding 4×10⁴ confinements per mm², or any other densitydescribed herein or equivalents thereof, wherein said opticalconfinements provide an effective observation volume that permitsobservation of individual molecules; and an optical system operativelycoupled to the optical confinements that detects signals from theeffective observation volume of said confinement; (b) mixing in theoptical confinements the plurality of target nucleic acid molecules,primers complementary to the target nucleic acid molecules,polymerization enzymes, and more than one type of nucleotides ornucleotide analogs to be incorporated into a plurality of nascentnucleotide strands, each strand being complementary to a respectivetarget nucleic acid molecule; (c) subjecting the mixture of step (b) toa polymerization reaction under conditions suitable for formation of thenascent nucleotide strands by template-directed polymerization of thenucleotides or nucleotide analogs; (d) illuminating the opticalconfinements with an incident light beam; and (e) identifying thenucleotides or the nucleotide analogs incorporated into the each nascentnucleotide strand.

The present invention also provides an apparatus comprising an array ofwaveguides on a solid support having a fill fraction greater than about0.0001, wherein said waveguides are suitable for holding a biologicalreagent, and wherein waveguides provide an effective observation volumethat permits observation of individual molecules present in saidbiological reagent; and an optical system that detects said individualmolecules in said waveguides, by e.g., detecting signals from theeffective observation volume. In one aspect, the array has a fillfraction greater than about 0.001. In another aspect, the array has afill fraction greater than about 0.01, in some instances greater than0.1, or within the range about 0.001 to about 0.1.

The present invention also provides various methods of using such highfill fraction array. In one embodiment, the present invention provides amethod of detecting a biological analyte. The method comprises opticallycapturing the analyte within an optical confinement that is created by(a) providing an array of waveguides having a fill fraction greater thanabout 0.0001; and (b) illuminating at least one waveguide within thearray that is suspected to contain the analyte with an incident lightbeam thereby detecting the analyte.

In another embodiment, the present invention provides a method ofperforming multiple chemical reactions involving a plurality of reactionsamples using the subject high fill fraction array. The method involves(a) providing a subject high fill fraction array; (b) placing theplurality of reaction samples comprising labeled reactants into thewaveguides in the array, wherein a separate reaction sample is placedinto a different waveguide in the array; (c) subjecting the array toconditions suitable for formation of products of the chemical reactions;and (d) detecting the formation of the products with an optical system.The step of detecting may comprise illuminating the different waveguideswith an incident light beam and detecting an optical signal emitted fromthe reaction samples. Applicable chemical reactions may involveprotein-protein interactions, nucleic acid-protein interactions, andnucleic acid-nucleic acid interactions. Specifically, the presentinvention provides a method of sequencing a plurality of target nucleicacid molecule using a fill fraction greater than about 0.0001.

The present invention further provides a method of sequencing nucleicacid using an array having a high fill faction. The method typicallyinvolves a) providing an array of waveguides having a fill fractiongreater than about 0.0001, or 0.001, or 0.01 or even 0.1; (b) mixing inthe waveguides the plurality of target nucleic acid molecules, primerscomplementary to the target nucleic acid molecules, polymerizationenzymes, and more than one type of nucleotides or nucleotide analogs tobe incorporated into a plurality of nascent nucleotide strands, eachstrand being complementary to a respective target nucleic acid molecule;(c) subjecting the mixture of step (b) to a polymerization reactionunder conditions suitable for formation of the nascent nucleotidestrands by template-directed polymerization of the nucleotides ornucleotide analogs; (d) illuminating the waveguides with an incidentlight beam; and (e) identifying the nucleotides or the nucleotideanalogs incorporated into the each nascent nucleotide strand.

Also included in the present invention is a redundant sequencing method.The method comprises (a) subjecting a target nucleic acid molecule to atemplate-directed polymerization reaction to yield a nascent nucleicacid strand that is complementary to the target nucleic acid molecule inthe presence of a plurality of types of nucleotides or nucleotideanalogs, and a polymerization enzyme exhibiting strand-displacementactivity; and (b) registering a time sequence of incorporation ofnucleotides or nucleotide analogs into the nascent nucleotide strand. Inone aspect of this embodiment, the target nucleic acid molecule is acircular nucleic acid, or is a linear or circular template strandsynthesized from a circular nucleic acid sequence such that thesynthesized strand includes multiple repeated copies of the originalcircular strand, and is thus is subject to the sequencing operations ofthe invention. In another aspect of this embodiment, the target nucleicacid molecule is sequenced multiple times, e.g., more than once, or morethan twice by the polymerization enzyme. In yet another aspect of thisembodiment, the polymerization enzyme is a DNA polymerase, such as amodified or unmodified Φ29 polymerase.

Further included in the present invention is a solid support having asurface wherein the surface has a polymerization enzyme array attachedto it, wherein members of the array comprise individually and opticallyresolved polymerization enzymes possessing strand-displacementactivities.

Also provided is a zero mode waveguide, comprising a first molecularcomplex immobilized therein, said molecular complex comprising apolymerization enzyme complexed with a target nucleic acid, wherein thepolymerization enzyme processes a sequence of nucleotides in said targetnucleic acid multiple times via template-dependent replication of thetarget nucleic acid.

Further provided by the present invention is a method of fabricating anarray of optical confinements that exhibits a minimal intensity ofdiffractive scattering of an incident wavelength. The method comprisesproviding a substrate; and forming the array of optical confinements onthe substrate such that individual confinements in the array areseparated from each other at a distance less than one half of thewavelength.

Finally, the present invention includes a method of fabricating anoptical confinement the method comprises a cladding surrounding a core,comprising: (a) providing a substrate coated with a layer ofphotoresist; (b) patterning said layer of photoresist to defineboundaries of said core; (c) removing said layer of photoresistsurrounding said defined boundaries so that a sufficient amount ofphotoresist remains to occupy said core; (d) depositing a layer ofcladding material over said remaining photoresist and said substrate;(e) removing at least a portion of said cladding material deposited oversaid remaining photoresist; and (f) removing said photoresist of step(e) to form said core surrounded by said cladding of said opticalconfinement. In one aspect, the photoresist is negative and saidpatterning step employs a positive pattern. In another aspect, thephotoresist is positive and said patterning step employs a negativepattern. The removing step can be effected by a technique selected fromthe group consisting of etching, mechanical polishing, ion milling, andsolvent dissolution. The layer of cladding material can be deposited bya thermal evaporation method or vapor deposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a top view of an array of illustrative opticalconfinements, here zero-mode waveguides arranged in a square format.

FIG. 2 depicts a top view of an array of illustrative opticalconfinements, here zero-mode waveguides arranged in a non-square format.

FIG. 3 depicts a top view of an illustrative 2-dimentional array with anillustrative angle and two different unit vector lengths.

FIG. 4 depicts a top view of an illustrative regular disposition ofZMWs.

FIG. 5 depicts an array of arrays, in which a subarray 71 is part of asuper array 72.

FIG. 6 illustrates a process of negative tone fabrication.

FIG. 7 illustrates an array of ZMWs optically linked to an opticalsystem.

FIG. 8 depicts a scanning electron micrographs of ZMW structuresfabricated by positive tone resist (left panels) or negative tone resist(right panels). The grain structure of the polycrystalline film isvisible in the image as flecks, and the ZMWs as dark round structures.

FIG. 9 depicts a single-molecule DNA sequence pattern recognition inZMWs using artificial pre-formed replication forks.

FIG. 10, depicts a coated ZMW 101 that is bound to a substrate 105. TheZMW comprises a sidewall 102, a coating 103 on the upper surface, and ametal film 104.

FIG. 11 depicts one alignment strategy and optical setup.

FIG. 12 depicts an alternative optical confinement made of porous film91 on a substrate 93. 92 represents the pores in the film.

FIGS. 13A-B depict an alignment detection system and the associatedcomponents.

FIG. 14 depicts several exemplary photocleavable blockers and theapplicable wavelength applied to cleave the blocking groups.

FIG. 15 depicts an exemplary reversible extension terminator in whichthe photocleavable blocker is conjugated to a detectable label (e.g.,fluorescent label).

FIG. 16 depicts an exemplary profile of fluorescent bursts correspondingto the time sequence of incorporation of two types of labelednucleotides or nucleotide analogs in single-molecule sequencing reactionusing the subject optical confinement.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of Integrated Circuit (IC) processingbiochemistry, chemistry, molecular biology, genomics and recombinantDNA, which are within the skill of the art. See, e.g., Stanley Wolf etal., SILICON PROCESSING FOR THE VLSI ERA, Vols 1-4 (Lattice Press);Michael Quirk et al., SEMICONDUCTOR MANUFACTURING TECHNOLOGY; Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd)edition (1989); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R.Taylor eds. (1995), all of which are incorporated herein by reference.

DEFINITIONS

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

“Luminescence” refers to the emission of light from a substance for anyreason other than a rise in its temperature. In general, atoms ormolecules emit photons of electromagnetic energy (e.g., light) when thenmove from an “excited state” to a lower energy state (usually the groundstate); this process is often referred to as “decay”. There are manycauses of excitation. If exciting cause is a photon, the luminescenceprocess is referred to as “photoluminescence”. If the exciting cause isan electron, the luminescence process is referred to as“electroluminescence”. More specifically, electroluminescence resultsfrom the direct injection and removal of electrons to form anelectron-hole pair, and subsequent recombination of the electron-holepair to emit a photon. Luminescence which results from a chemicalreaction is usually referred to as “chemiluminescence”. Luminescenceproduced by a living organism is usually referred to as“bioluminescence”. If photoluminescence is the result of a spin allowedtransition (e.g., a single-singlet transition, triplet-triplettransition), the photoluminescence process is usually referred to as“fluorescence”. Typically, fluorescence emissions do not persist afterthe exciting cause is removed as a result of short-lived excited stateswhich may rapidly relax through such spin allowed transitions. Ifphotoluminescence is the result of a spin forbidden transition (e.g., atriplet-singlet transition), the photoluminescence process is usuallyreferred to as “phosphorescence”. Typically, phosphorescence emissionspersist long after the exciting cause is removed as a result oflong-lived excited states which may relax only through suchspin-forbidden transitions. A “luminescent label” or “luminescentsignal” may have any one of the above-described properties.

The term “electromagnetic radiation” refers to electromagnetic waves ofenergy including, for example, in an ascending order of frequency (oralternatively, in a descending order of wavelength), infrared radiation,visible light, ultraviolet (UV) light, X-rays, and gamma rays.

As used herein, an “effective observation volume” typically refers tothat volume that is observable by the detection means employed for agiven application. For example, in the case of fluorescence baseddetection, it is that volume which is exposed to excitation radiationand/or from which emission radiation is gathered by an adjacent opticaltrain/detector. By way of example, in the case of a zero mode waveguideused for certain applications, an effective observation volume isdictated by the propagation of excitation radiation into the waveguidecore, and particularly that volume that is exposed to light that is atleast 1%, and preferably at least 10% of the original intensity ofexcitation radiation entering the waveguide core. Such intensities andvolumes are readily calculable from the particular conditions of theapplication in question, including the wavelength of the excitationradiation and the dimensions of the waveguide core (See, e.g., U.S. Pat.No. 6,917,726, incorporated herein by reference in its entirety for allpurposes).

A “primer” is a short polynucleotide, generally with a free 3′ OH group,that binds to a target nucleic acid (or template) potentially present ina sample of interest by hybridizing with the target nucleic acid, andthereafter promoting polymerization of a polynucleotide complementary tothe target.

The terms “operatively linked to” or “operatively coupled to” are usedinterchangeably herein. They refer to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner.

The term “nucleotide” generally refers to a molecule comprising a base,sugar and one or more anionic groups, preferably phosphates. Themolecule may comprise one, two, three, four, five or more phosphatesgroups and/or other groups such as sulfate. The term also encompassesnucleotide analogs that are structurally analogous to naturallyoccurring nucleotides and are capable of acting substantially likenucleotides, for example exhibiting base complementarity with one ormore of the bases that occur in DNA or RNA, and/or being capable ofbase-complementary incorporation in synthesizing nucleotide strand by apolymerization enzyme.

The term “polynucleotide” refers to a polymeric form of “nucleotides” ofany length.

A “type of nucleotide” refers to a set of nucleotides that share acommon characteristic that is to be detected. For instance, the types ofnucleotides can be classified into four categories: A, T, C, and G forDNA, or A, U, C and G for RNA. In some embodiments, each type ofnucleotides used in the a reaction will be labeled with a unique labelthat is distinguishable from the rest.

The term “optical confinement” refers to an area in which the reactantsfor an intended reaction within the confinement are confined andresolved by optical means.

A “polynucleotide probe” refers to a polynucleotide used for detectingor identifying its corresponding target polynucleotide in ahybridization reaction.

The term “hybridize” as applied to a polynucleotide refers to theability of the polynucleotide to form a complex that is stabilized viahydrogen bonding between the bases of the nucleotide residues in ahybridization reaction. The hydrogen bonding may occur by Watson-Crickbase pairing, Hoogstein binding, or in any other sequence specificmanner. The complex may comprise two strands forming a duplex structure,three or more strands forming a multi-stranded complex, a singleself-hybridizing strand, or any combination of these. The hybridizationreaction may constitute a step in a more extensive process, such as theinitiation of a PCR reaction, or the enzymatic cleavage of apolynucleotide by a ribozyme.

Hybridization can be performed under conditions of different“stringency”. Relevant conditions include temperature, ionic strength,time of incubation, the presence of additional solutes in the reactionmixture such as formamide, and the washing procedure. Higher stringencyconditions are those conditions, such as higher temperature and lowersodium ion concentration, which require higher minimum complementaritybetween hybridizing elements for a stable hybridization complex to form.In general, a low stringency hybridization reaction is carried out atabout 40° C. in 10×SSC or a solution of equivalent ionicstrength/temperature. A moderate stringency hybridization is typicallyperformed at about 50° C. in 6×SSC, and a high stringency hybridizationreaction is generally performed at about 60° C. in 1×SSC.

When hybridization occurs in an antiparallel configuration between twosingle stranded polynucleotides, the reaction is called “annealing” andthose polynucleotides are described as “complementary”. Adouble-stranded polynucleotide can be “complementary” or “homologous” toanother polynucleotide, if hybridization can occur between one of thestrands of the first polynucleotide and the second. “Complementarity” or“homology” (the degree that one polynucleotide is complementary withanother) is quantifiable in terms of the proportion of bases in opposingstrands that are expected to form hydrogen bonding with each other,according to generally accepted base pairing rules.

Structure of the Optical Confinements of the Present Invention

One aspect of the present invention is the design of optical devices andmethods for characterizing molecules and/or monitoring chemicalreactions. The optical devices of the present invention allowmultiplexing of large numbers of single-molecule analyses underphysiologically relevant conditions.

In one embodiment, the present invention provides a high density arrayof optical confinements having a surface density exceeding 4×10⁴confinements per mm², preferably exceeding 10⁵, wherein the individualconfinement in the array provides an effective observation volume on theorder of zeptoliters. The array may contain at least about 2×10⁵, atleast about 10⁶, or at least about 10⁷ optical confinements. Preferably,the individual confinement in the array provides an effectiveobservation volume less than about 1000 zeptoliters, more preferablyless than about 900, more preferably less than about 80, even morepreferably less than about 10 zeptoliters. Where desired, an effectiveobservation volume less than 1 zeptoliter can be provided. In apreferred aspect, the individual confinement yields an effectiveobservation volume that permits resolution of individual moleculespresent at a physiologically relevant concentration. The physiologicallyrelevant concentrations for most biochemical reactions range frommicro-molar to millimolar because most of the enzymes have theirMichaelis constants in these ranges. Accordingly, preferred array ofoptical confinements has an effective observation volume for detectingindividual molecules present at a concentration higher than about 1micromolar (μM), or more preferably higher than 50 μM, or even higherthan 100 μM.

To achieve the required observation volume for single-molecule analysisunder physiologically relevant conditions, the array may comprisezero-mode waveguides or alternative nanoscale optical structures. Suchalternative structures include but are not limited to porous films withreflective index media, and confinements using index matching solids.

As used herein, “zero-mode waveguide” refers to an optical guide inwhich the majority of incident radiation is attenuated, preferably morethan 80%, more preferably more than 90%, even more preferably more than99% of the incident radiation is attenuated. As such high level ofattenuation, no significant propagating modes of electromagneticradiation exist in the guide. Consequently, the rapid decay of incidentelectromagnetic radiation at the entrance of such guide provides anextremely small observation volume effective to detect single molecules,even when they are present at a concentration as high as in themicromolar range.

The zero-mode waveguide of the present invention typically comprises acladding surrounding a core (i.e., partially or fully), wherein thecladding is configured to preclude propagation of electromagnetic energyof a wavelength higher than the cutoff wavelength longitudinally throughthe core of the zero-mode waveguide. The cladding is typically made ofmaterials that prevent any significant penetration of the electric andthe magnetic fields of an electromagnetic radiation. Suitable materialsfor fabricating the cladding include but are not limited to alloys,metals, and semi-conducting materials, and any combination thereof.Alloys include any of the numerous substances having metallic propertiesbut comprising two or more elements of which at lest one is a metal.Alloys may vary in the content or the amount of the respectiveelements-whether metallic or non metallic. Preferred alloys generallyimprove some desirable characteristics of the material over a pureelemental material. Characteristics that can be improved through the useof mixtures of materials include, chemical resistance, thermalconductivity, electrical conductivity, reflectivity, grain size,coefficient of thermal expansion, brittleness, temperature tolerance,conductivity, and/or reduce grain size of the cladding.

In general, alloys suitable for the present invention may involvemixtures where one component is present at fractions as low as 0.0001%.In other instances, alloys with large fractions of more than onecompound will be desirable. One embodiment of the ZMW uses aluminum asthe cladding of the ZMW structure. As an example of how alloys can bebeneficial to a ZMW structure, it is useful to consider different alloysof aluminum in how they would affect a ZMW. In the art of metalurgy,numerous materials are alloyed with aluminum. Non-limiting examples ofmaterials suitable to alloy with aluminum are antimony, arsenic,beryllium, bismuth, boron, cadmium, calcium, carbon, cerium, chromium,cobalt, copper, gallium, hydrogen, indium, iron, lead, lithium,magnesium, manganese, mercury, molybdenum, nickel, niobium, phosphorous,silicon, vanadium, zinc and others. By way of example of how theintroduction of another element could beneficially impact the ZMWperformance, the introduction of boron to aluminum is known to increasethe conductivity of aluminum. An increase in conductivity of the metalfilm may improve the performance by decreasing the penetration depththereby decreasing the observation volume. A preferred embodimentincludes an alloy of aluminum that is more than 0.0001% of a dopant. Amore preferred embodiment includes an alloy of aluminum that is morethan 0.005% of a dopant. A still more preferred embodiment includes anallow of aluminum that is more than 0.1% of a dopant.

In contrast, some materials are expected to decrease the performance ofthe ZMW structure, and in these instances it will be desirable to takemeasures to eliminate certain impurities. For example, in certainapplications it may be desirable to decrease the amount of lead orarsenic if toxicity of the device is a concern. A preferred embodimentof the device includes a metal film that is less than 1% arsenic. A morepreferred embodiment of the device includes a metal films that is lessthan 0.1% arsenic. A still more preferred embodiment includes a metalfilm that is less than 0.001% arsenic. A still more preferred embodimentincludes a metal film that is less than 0.00001% arsenic. An additionalpreferred embodiment includes a metal film that is less than 1% lead. Astill more preferred embodiment includes a metal film that is less than0.1% lead. A still more preferred embodiment includes a metal film thatis less than 0.01% lead. A still more preferred embodiment includes ametal film that is less than 0.001% lead. A still more preferredembodiment includes a film that is less than 0.00001% lead. In otherapplications where optical.confinement performance is especiallyimportant, impurities that tend to reduce the conductivity, therebyworsening the confinement, will be undesirable. For example, vanadium isknown in the art of metallurgy to reduce the conductivity of aluminum. Apreferred embodiment includes a metal film that is less than 0.1%vanadium. A still more preferred embodiment includes a metal film thatis less than 0.01% vanadium. A still more preferred embodiment includesa film that is less than 0.001% vanadium.

Semi-conducting materials suitable for fabricating the cladding aregenerally opaque, and they include silicon, silicates, silicon nitride,gallium phosphide, gallium arsenide, or any combinations thereof.

The cladding of the subject zero-mode waveguide may be coated withmaterials to improve the surface quality. For instance, coating mayenhance the durability of the cladding material. In addition, coating isparticularly desirable if the reactants contained in the core are proneto interact or adhere to the cladding material. A variety of appropriatecoating materials are available in the art. Some of the materials maycovalently adhere to the surface, others may attach to the surface vianon-covalent interactions. Non-limiting examples of coating materialsinclude aluminum oxide film, silanization reagent such asdimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane ortrimethylchlorosilane, polymaleimide, and siliconizing reagents such assilicon oxide, Aquasil™, and Surfasil™. An illustrative coated ZMW (101)is shown in FIG. 10. The ZMW (101) is bound to a substrate 105. The ZMWcomprises a sidewall 102, a coating 103 on the upper surface, and ametal film 104.

In certain embodiments, it may be advantageous to construct theconfinement from metal compositions that are inhomogeneous combinationsof more than one material. For example, for certain applications, it maybe beneficial to provide a composition that comprises more than onelayer, each layer having a different composition, or composition thatvaries within a layer. This can have beneficial effects on severalaspects of the performance of the confinement, including but not limitedto the nature of the optical confinement, the structural strength andbehavior of the device, the characteristics of the surface chemistry ofthe device or the like. In one embodiment the confinement comprises twolayers in which one of the layers serves to enhance the adhesion of thesecond layer to a substrate. In another embodiment, the composition ofthe cladding film varies as a function of the axial position relative tothe confinement, so as to provide different optical performance thanwould be obtained from a layer of uniform composition. In a particularversion of this embodiment, the film comprises a composition that has alarger value of skin depth close to the surface of the substrate, andcomprises a composition that has a smaller value of skin depth fartherfrom the surface of the substrate, so that the nature of the confinementis to be more uniform in shape near the surface and then tapering offmore quickly a larger distances away from the substrate. In anotherembodiment, the thicknesses of two different layers comprising thecladding of the confinement are chosen so that a specific opticalcondition is achieved at the substrate of the device, such asconstructive or destructive interference.

The internal cavity (i.e., the core) surrounded by the cladding mayadopt a convenient size, shape or volume so long as propagating modes ofelectromagnetic radiation in the guide is effectively prevented. Thecore typically has a lateral dimension less than the cutoff wavelength(λ_(c)). For a circular guide of diameter d and having a clad of perfectconductor, λ_(c) is approximately 1.7×d. The cross sectional area of thecore may be circular, elliptical, oval, conical, rectangular,triangular, polyhedral, or in any other shape. The various shapes canhave particular suitability for certain applications. For instance,elongated cross-sections can be useful to provide enhanced access tomolecules with mechanical persistence or stiffness, such as DNA. Crosssections ranging from extended slots to ovals of various aspect ratiowill significant increase the accessibility of the persistent moleculeto the detection zone of the structure, without excessive compromise inthe axial attenuation of radiation. Although uniform cross sectionalarea is preferred, the cross sectional area may vary at any given depthof the guide if desired. Preferred average cross sectional areas rangefrom 100 nm² to 10,000 nm².

In a preferred embodiment, the core is non-cylindrical. In one aspect ofthis embodiment, a non-cylindrical core comprises an opening on theupper surface and a base at the bottom surface that is entirelysurrounded by the cladding, wherein the opening is narrower in lateraldimension than the base. This configuration significantly restricts thediffusion of reactants, and hence increases the average residence timein the observation volume. Such configuration is particularly useful formeasuring the association rate constant (on-rate) of a chemicalreaction. In another aspect, the core comprises an opening that is widerin lateral dimension than the base. Such configuration allows easieraccess to large molecules that impose a steric or entropic hindrance toentering the structure if the open end of the zero mode waveguide was assmall as the base needed to be for optical performance reasons. Examplesinclude the accessibility for long strand polyelectrolytes such as DNAmolecules that are subject to entropic forces opposing entry into smallopenings.

The zero-mode waveguides embodied in the present invention have arelatively high fill fraction ratio, typically above 0.0001, preferablyabove 0.001, more preferably above 0.01, and even more preferably above0.1. As used herein, “fill fraction” of a pattern refers to the ratio ofthe area occupied by the foreground of the pattern to the total areaoccupied by the pattern (foreground and background, together). The terms“fill fraction ratio” and “fill faction” are used interchangeably. Inthe context of zero-mode waveguide, the foreground is considered to bethe area occupied by the core of the zero-mode waveguide, and thebackground is the area between the zero-mode waveguide (e.g., thealuminum film that forms the cladding in certain designs). The zero-modewaveguides with high fill fraction ratios are particularly useful forperforming homogenous assays. The fill fraction can be calculated bysumming the total areas of all of the zero-mode waveguides in the arrayand dividing by the total available area including both the zero-modewaveguides and the spaces between them. For example, if a zero-modewaveguide has a diameter of 50 nm, then the area of this zero-modewaveguide is one fourth of 7,850 square nanometers or 1962.5 nm². Ifthese zero-mode waveguides are in a square array separated by 100 nm,the total available area is 10,000 square nanometers for each zero-modewaveguide. Therefore, the array has a fill fraction of one fourth of 78%or 19.6%, which would provide nearly four orders of magnitude highersignal strength in a surface binding assay than a zero-mode waveguidehaving a fill fraction on the order of 0.01%.

In a bioassay such as an ELISA or other molecular binding bioassay, onelimitation is the inability to operate “homogeneously”, or in a modewhere solutions may be added to a mixture but nothing removed. Thiscomplicates highly multiplexed assays, as provisions for both adding andremoving material from a large number of wells is significantly morecomplex than the provisions for simply adding materials. In the case ofthe ELISA assay, the removal of materials is necessary, because thefluorescent (or other) markers that remain free in solution at the endof the assay would interfere with the ability to detect markers bound tothe reaction surface. Techniques to overcome this have been devised toexploit the short range of radioactive emissions from certainradioisotopes, but these techniques have inherent difficultiesassociated with personnel safety and waste disposal. Other methods forconfining the sensitivity of the assay to the surface have been devised,such as total internal reflection confinement (TIR), and confocaldetection. The zero-mode waveguide photonic structure allows a simplerand less expensive optical system configuration than either of thesetechniques, and vastly outperforms both from the perspective ofconfinement of sensitivity to the surface.

The fill fraction is important in bioassays, because the effective probearea is limited to the surface area of the bottoms of the zero-modewaveguide in the detection region. The amount of signal detectable insuch an assay will be directly proportional to the available area, andhaving a larger fraction of the available surface occupied by zero-modewaveguides will thus increase the signal strength of measurements ofsuch assays. A high fill fraction structure would be generally useful inany surface sensitivity application, not limited to the ELISA assay.

The cutoff wavelength is the wavelength above which the waveguide isessentially incapable of propagating electromagnetic energy along thewaveguide under the illumination geometry used. Given the geometry ofthe core, and the properties of the cladding material, as well as thewavelength of the incident electromagnetic radiation, one skilled in theart can readily derive the cutoff wavelength by solving the Maxwell'sequations (see, e.g., John D. Jackson, CLASSICAL ELECTRODYNAMICS, secondedition, John Willey and Sons). The choice of the incident wavelengthwill depend on the particular application in which the subject array isto be employed. In certain aspects, the incident wavelength may beselected from a range of about 10 nm to about 1 mm. For detectingfluorescent signals, the incident wavelength is typically selected fromthe range of about 380 nm to about 800 nm. Polarized (linearly orpreferably circularly polarized) or unpolarized incident radiation isgenerally employed to illuminate the array in order to create a desiredobservation volume.

In a separate embodiment, the present invention provides an alternativeoptical confinement termed external reflection confinement (ERC). Incontrast to the conventional total internal reflection confinement(IRC), the low index medium is the electromagnetic radiation carrier,and the high index (and opaque) medium is the reflector. As such, theroles of the refractive indices are reversed as compared to the IRCsituation. ERC generally requires some kind of means to provide theanalyte (ie., the molecules under investigation) in the opaque phase.

IRC relies on reflection of an electromagnetic radiation incident on aninterface between high index of refraction and low index of refraction.When light is incident above the critical angle of total internalreflection (known in the art), all of the incident electromagneticradiation is reflected and none is transmitted into the low index phase.A thin region of evanescent radiation is established proximal to theinterface on the low index side. This radiation field is typically anexponentially decaying field with an attenuation length in the rangefrom about 100 nm to about 200 nm, depending on the angle of incidenceand the indices of refraction of the two phases. If the low index phaseis a solution containing an analyte, then the evanescent radiation canbe used to probe the analyte in the solution with a high degree ofsurface sensitivity.

In ERC, the carrier of the propagating electromagnetic radiation is atransparent low index film, and the analyte-bearing medium is ahigh-index metallic opaque film. In this case, most of the radiation isreflected irrespective of the angle of incidence, and non-reflectedlight is rapidly attenuated according to the skin depth of the metal.Typically, means is provided to convey the analyte within the metalphase. Theses means can take the form of a nanocapillary tubeconstructed within the metal layer. When sufficiently small, thepresence of such a tube will have little effect on the distribution ofenergy in the two media, but can be amply large enough to conveybiomolecules. To be small enough, any defects in the metal film must besmall compared with the wavelength of the illumination. This can beachieved because of the large ratio between the wavelength of visiblelight, and the typical size of biomolecules of interest. While visiblelight is typically between 400 nm and 750 nm in wavelength, biomoleculesof interest are generally in the vicinity of 1-30 nm in diameter. Theattenuation of the radiation at the interface can be used to confineillumination to a very small region of the analyte. A small hole in anindex matched (to water) film on a high index substrate could providelateral confinement beyond what is possible with diffraction limitedoptics in the TIR context. This could give 100 zeptoliter confinement inprinciple. In this method, a version of total internal reflectionconfinement is used in which a solid material index-matched to theanalyte solution is applied to the substrate surface and then perforatedwith nanoscale holes. When used in TIR mode, these structures willprovide additional confinements above what can be obtained with TIRalone.

Other alternative confinements are index matching solids. As anillustrative example, such optical confinement can be fabricatedstarting with a high index transparent susbtrate such as sapphire, spincoat 200 nm of PMMA (polymethyl methacrylate) resist resin. Exposure toelectron beam lithography will render isolated spots soluble accordingto the pattern applied. After development, the device will havenano-scale holes in the PMMA layer and are ready to be used in a TIRsetup. Axial confinement is unaffected by the PMMA layer, as it hasnearly the same index of refraction as the solution containing theanalyte, but the solution is physically prevented from approaching nearthe surface except where the holes are situated, providing a degree oflateral confinement given by the diameter of the holes.

The optical confinements can be provided with an optical system capableof detecting and/or monitoring interactions between reactants at thesingle-molecule level. Such optical system achieves these functions byfirst generating and transmitting an incident wavelength to thereactants contained in the confinements, followed by collecting andanalyzing the optical signals from the reactants. Such systems typicallyemploy an optical train that directs signals from an array ofconfinements onto different locations of an array-based detector tosimultaneously detect multiple different optical signals from each ofmultiple different confinements. In particular, the optical trainstypically include optical gratings or wedge prisms to simultaneouslydirect and separate signals having differing spectral characteristicsfrom each confinement in an array to different locations on an arraybased detector, e.g., a CCD. By separately directing signals from eachconfinement to different locations on a detector, and additionallyseparating the component signals from each confinement to separatelocations, one can simultaneously monitor multiple confinements, andmultiple signals from each confinement.

The optical system applicable for the present invention comprises atleast two elements, namely an excitation source and a photon detector.The excitation source generates and transmits incident light used tooptically excite the reactants contained in the optical confinement.Depending on the intended application, the source of the incident lightcan be a laser, laser diode, a light-emitting diode (LED), aultra-violet light bulb, and/or a white light source. Where desired,more than one source can be employed simultaneously. The use of multiplesources is particularly desirable in applications that employ multipledifferent reagent compounds having differing excitation spectra,consequently allowing detection of more than one fluorescent signal totrack the interactions of more than one or one type of moleculessimultaneously. A wide variety of photon detectors are available in theart. Representative detectors include but are not limited to opticalreader, high-efficiency photon detection system, photodiode (e.g.avalanche photo diodes (APD)), camera, charge couple device (CCD),electron-multiplying charge-coupled device (EMCCD), intensified chargecoupled device (ICCD), and confocal microscope equipped with any of theforegoing detectors. Where desired, the subject arrays of opticalconfinements contain various alignment aides or keys to facilitate aproper spatial placement of the optical confinement and the excitationsources, the photon detectors, or the optical transmission element asdescribed below.

The subject optical system may also include an optical transmissionelement whose function can be manifold. First, it collects and/ordirects the incident wavelength to the optical confinement containingthe reactants. Second, it transmits and/or directs the optical signalsemitted from the reactants inside the optical confinement to the photondetector. Third, it may select and/or modify the optical properties ofthe incident wavelengths or the emitted wavelengths from the reactants.Illustrative examples of such element are diffraction gratings, arrayedwaveguide gratings (AWG), optic fibers, optical switches, mirrors,lenses (including microlens and nanolens), collimators. Other examplesinclude optical attenuators, polarization filters (e.g., dichroicfilter), wavelength filters (low-pass, band-pass, or high-pass),wave-plates, and delay lines. In some embodiments, the opticaltransmission element can be planar waveguides in optical communicationwith the arrayed optical confinements. For instance, a planar waveguidescan be operatively coupled to an array of zero-mode waveguides todirectly channel incident wavelengths to the respective cores of thezero-mode waveguides so as to minimize the loss of wave energy. Theplanar channel can be included as a detachable unit located at the baseof array substrate, or it can be bonded to the substrate as an integralpart of the array.

The optical transmission element suitable for use in the presentinvention encompasses a variety of optical devices that channel lightfrom one location to another in either an altered or unaltered state.Non-limiting examples of such optical transmission devices includeoptical fibers, diffraction gratings, arrayed waveguide gratings (AWG),optical switches, mirrors, (including dichroic mirrors), lenses(including microlens and nanolens), collimators, filters, prisms, andany other devices that guide the transmission of light through properrefractive indices and geometries.

In a preferred embodiment, the optical confinement of the presentinvention is operatively coupled to a photon detector. For instance, thearrayed optical confinement is operatively coupled to a respective andseparate photon detector. The confinement and the respective detectorcan be spatially aligned (e.g., 1:1 mapping) to permit an efficientcollection of optical signals from the waveguide. A particularlypreferred setup comprises an array of zero-mode waveguides, wherein eachof the individual waveguides is operatively coupled to a respectivemicrolens or a nanolens, preferably spatially aligned to optimize thesignal collection efficiency. Alternatively, a combination of anobjective lens, a spectral filter set or prism for resolving signals ofdifferent wavelengths, and an imaging lens can be used in an opticaltrain, to direct optical signals from each confinement to an arraydetector, e.g., a CCD, and concurrently separate signals from eachdifferent confinement into multiple constituent signal elements, e.g.,different wavelength spectra, that correspond to different reactionevents occurring within each confinement.

An exemplary optical setup is shown in FIG. 7, in which an array of ZMWsis optically linked to an optical system. This system comprises a ZMWarray film (81), a glass cover slip (82) through which light transmitsand further converges through set of integral lenses (83) made of amaterial having a different index of refraction than that of the glass.In particular, 84 shows a ZMW structure, 85 indicates a ray of lightbeing focused onto the ZMW by the integral lenses such as the embeddedmicrolens.

FIG. 11 depicts one alignment strategy and optical system. The systemcomprises a photodetector 131, an optional lens 132 for collectinglight, a ZMW 133 having a metal film 134 coupled to a substrate 135, andan objective lens 136 that is aligned with the incident light beam 137.FIG. 13 depicts an exemplary alignment detection system and theassociated components. The illustrative system 13A comprises an opticalconfinement such as a zero-mode waveguide 111 having a metal film 113coupled to a substrate 114. The zero-mode waveguide 111 typicallycontains signal generating molecules 112, and is optically linked to theassociated components including an objective lens 115, a beamsplitter/dichroic cube 117, optically a telen lens 120 (used in infinitycorrected systems), and a photodetector 122 (e.g., a quadrantphotodetector). 116 depicts rays propagating though system. 118 depictsthe incident illumination rays. 119 depicts the return rays movingtowards the detector 122. FIG. 13B depicts a front view of the quadrantphotodiode. Shown in the center of the figure is a beam mis-aligned onthe center of the quadrant detector. The four voltages generated by thefour quadrants can be processed to determine the degree and direction ofmis-alignment of the beam and thus the optical confinement such as ZMW111.

The subject arrays may comprise a single row or a plurality of rows ofoptical confinements on the surface of a substrate, where a plurality oflanes are present, for example, usually at least 2, more commonly morethan 10, and more commonly more than 100. The subject array of opticalconfinements may align horizontally or diagonally long the x-axis or they-axis of the substrate. The individual confinements can be arrayed inany format across or over the surface of the substrate, such as in rowsand columns so as to form a grid, or to form a circular, elliptical,oval, conical, rectangular, triangular, or polyhedral pattern. Tominimize the nearest-neighbor distance between adjacent opticalconfinements, a hexagonal array is preferred.

The array of optical confinements may be incorporated into a structurethat provides for ease of analysis, high throughput, or otheradvantages, such as in a microtiter plate and the like. Such setup isalso referred to herein as an “array of arrays.” For example, thesubject arrays can be incorporated into another array such as microtiteror multi-well plate wherein each micro well of the plate contains asubject array of optical confinements. Typically, such multi-well platescomprise multiple reaction vessels or wells, e.g., in a 48 well, 96well, 384 well or 1536 well format. In such cases, the wells aretypically disposed on 18 mm, 9 mm, 4.5 mm, or 2.25 mm centers,respectively.

An illustrative array of arrays is depicted in FIG. 5 in which asubarray 71 is part of a super array 72. Arrays can also be arranged inlattices. For example, FIG. 4 depicts a top view of an illustrativeregular disposition of ZMWs. In this configuration, there is a latticedefined by the parameters d1, d2, and the angle 53. In addition to a ZMWat each lattice point, there is a complex unit cell that comprises aplurality of ZMWs in an arrangement that is defined by a list of anglesand distances with one angle and one distance for each element of theunit cell. In particular, 52 represents the first lattice distance, 53represents the lattice angle, 54 represents the second lattice distance,55 represents the unit cell first distance, and 56 represents unit cellfirst angle. While this figure shows an array with a unit cell of twocomponents, the unit cell can have any plurality of elements.

As described above, the subject arrays comprise a plurality of opticalconfinements. In some embodiments, the arrays have at least about 20×10⁴distinct optical confinements, preferably at least about 20×10⁶ distinctconfinements, and more preferably at least about 20×10⁸ confinements.The density of the spots on the solid surface in certain embodiments isat least above 4×10⁴ confinements per mm², and usually at least about8×10⁴, at least about 1.2×10⁵, or at least about 4×10⁶ confinements permm², but does not exceed 4×10¹² confinements per mm², and usually doesnot exceed about 4×10¹⁰ confinements per mm². The overall size of thearray generally ranges from a few nanometers to a few millimeters inthickness, and from a few millimeters to 50 centimeters in width orlength. Preferred arrays have an overall size of about few hundredmicrons in thickness and may have any width or length depending on thenumber of optical confinements desired.

In one example as shown in FIG. 1, the array of optical confinements,e.g. zero-mode waveguides, are arranged in a square format. The arraycomprises a representative zero-mode waveguide 21, separated from anadjacent waveguide by a distance “d” (22 represents the inter-zero modewaveguide spacing). In another example as shown in FIG. 2, the array ofoptical confinements, e.g. zero-mode waveguides, are arranged in anon-square format. The array comprises a representative zero-modewaveguide 31, separated from an adjacent waveguide by a distance “d” (32represents the inter-zero mode waveguide spacing). 33 shows the angleformed between any three adjacent ZMWs (e.g., 60 degrees). FIG. 3depicts a top view of another illustrative 2-dimentional array. Theadjacent optical confinements are separated in one dimension by adistance of “d1” and in another dimension by a distance of “d2”, with aunit vector angle 43.

The spacing between the individual confinements can be adjusted tosupport the particular application in which the subject array is to beemployed. For instance, if the intended application requires adark-field illumination of the array without or with a low level ofdiffractive scattering of incident wavelength from the opticalconfinements, then the individual confinements are typically placedclose to each other relative to the incident wavelength.

Accordingly, in one aspect, the present invention provides an array ofzero-mode waveguides comprising at least a first and at least a secondzero-mode waveguide, wherein the first zero-mode waveguide is separatedfrom the second zero-mode waveguide by a distance such that uponillumination with an incident wavelength, intensity of diffractivescattering observed from the first zero-mode waveguide at a given angleis less than that if the first zero-mode waveguide were illuminated withthe same incident wavelength in the absence of the second zero-modewaveguide. Diffractive scattering can be reduced or significantlyeliminated if an array comprises zero-mode waveguides spaced in aregular spaced lattice where the separation of zero-mode waveguides fromtheir nearest neighbors is less than half the wavelength of the incidentwavelength. In this regime, the structure behaves as a zero-ordergrating. Such gratings are incapable of scattering incident lightdespite having a large number of elements that by themselves wouldscatter very effectively. This arrangement is highly desirable forillumination approaches such as dark field illumination, where surfacescattering would cause excitation radiation to be collected by theobjective lens, thus increasing background noise. Useful wavelengths forillumination range from 250 nm up to 8 microns, meaning that an array ofzero-mode waveguides with a spacing of less than 4000 nm would still beuseful for application in this manner. A spacing of less than 2000 nm ismore preferable, while a spacing of less than 1000 nm is even morepreferable in this respect. Some configurations with spacing larger thanone half of the wavelength can have the same advantage if theillumination is applied asymmetrically, or if the collection cone angleis configured to be less than 90 degrees. In addition to the benefit ofreduced diffractive scattering, narrow spacing between the individualconfinements decreases the illumination area and thus lowers the powerdemand.

Arrays having the optical confinements spaced far apart relative to theincident wavelength also have desirable properties. While theangle-dependent scattering raises the background signal that could bedisadvantageous for certain applications, it provides a meansparticularly suited for characterizing the size and shape of the opticalconfinements. It also readily permits ensemble bulk measurements ofmolecule interactions, involving especially unlabelled molecules. Arrayssuited for such applications generally contain individual confinementsseparated by more than one wavelength of the incident radiation, usuallymore than 1.5 times the incident wavelength, but usually does not exceed150 times the incident wavelength.

Kits:

The present invention also encompasses kits containing the opticalconfinement arrays of this invention. Kits embodied by this inventioninclude those that allow characterizing molecules and/or monitoringchemical reactions at a single-molecule level. Each kit usuallycomprises the devices and reagents which render such characterizationand/or monitoring procedure possible. Depending on the intended use ofthe kit, the contents and packaging of the kit will differ. Where thekit is for DNA sequencing, the kit typically comprises: (a) an array ofoptical confinements, preferably zero-mode waveguides of the presentinvention, that permits resolution of individual molecules or thereaction of individual molecules, such as those that are present at aconcentration higher than about 1 micromolar; (b) sequencing reagentstypically including polymerases, aqueous buffers, salts, primers, andnucleotides or nucleotide analogs. Where desired a, ‘control’ nucleicacids of known sequence can be included to monitor the accuracy orprogress of the reaction.

The reagents can be supplied in a solid form, immobilized form, and/ordissolved/suspended in a liquid buffer suitable for inventory storage,and later for exchange or addition into the reaction medium when thetest is performed. Suitable individual packaging is normally provided.The kit can optionally provide additional components that are useful inthe procedure. These optional components include, but are not limitedto, buffers, capture reagents, developing reagents, labels, reactingsurfaces, control samples, instructions, and interpretive information.Diagnostic or prognostic procedures using the kits of this invention canbe performed by clinical laboratories, experimental laboratories,practitioners, or private individuals.

Preparation of the Optical Confinements:

The array of the present invention can be manufactured usingnanofabrication techniques provided by the present invention, as well asthose known in the fields of Integrated Circuit (IC) andMicro-Electro-Mechanical System (MEMS). The fabrication processtypically proceeds with selecting an array substrate, followed by usingappropriate IC processing methods and/or MEMS micromachining techniquesto construct and integrate the optical confinement and other associatedcomponents:

Array Substrate:

In some embodiments, the array of optical confinements is present on arigid substrate. In other embodiments concerning, e.g., porous filmswith reflective index media, flexible materials can be employed. Ingeneral, a rigid support does not readily bend. Examples of solidmaterials which are not rigid supports with respect to the presentinvention include membranes, flexible metal or plastic films, and thelike. As such, the rigid substrates of the subject arrays are sufficientto provide physical support and structure to optical confinementspresent thereon or therein under the assay conditions in which the arrayis employed, particularly under high throughput handling conditions.

The substrates upon which the subject patterns of arrays are disposed,may take a variety of configurations ranging from simple to complex,depending on the intended use of the array. Thus, the substrate couldhave an overall slide or plate configuration, such as a rectangular ordisc configuration, where an overall rectangular configuration, as foundin standard microtiter plates and microscope slides, is preferred.Generally, the thickness of the rigid substrates will be at least about0.01 mm and may be as great as 1 cm or more, but will usually not exceedabout 5 cm. Both the length and the width of rigid substrate will varydepending on the size of the array of optical confinements that are tobe fabricated thereon or therein.

The substrates of the subject arrays may be fabricated from a variety ofmaterials. The materials from which the substrate is fabricated ispreferably transparent to visible and/or UV light. Suitable materialsinclude glass, semiconductors (e.g., silicate, silicon, silicates,silicon nitride, silicon dioxide, quartz, fused silica, and galliumarsenide), plastics, and other organic polymeric materials. In preferredaspects, silica based substrates like glass, quartz and fused silica areused as the underlying transparent substrate material.

The substrate of the subject arrays comprise at least one surface onwhich a pattern of optical confinements is present, where the surfacemay be smooth or substantially planar, or have irregularities, such asdepressions or elevations. The surface may be modified with one or moredifferent layers of compounds that serve to modulate the properties ofthe surface in a desirable manner. Modification layers of interestinclude: inorganic and organic layers such as metals, metal oxides,polymers, small organic molecules, functional moieties such asavidin/biotin and the like. The choice of methods for applying thecoating materials will depend on the type of coating materials that isused. In general, coating is carried out by directly applying thematerials to the zero-mode waveguide followed by washing the excessiveunbound coating material from the surface. Alternatively oradditionally, coating materials may be deposited using otherconventional techniques, such as chemical vapor deposition (CVD),sputtering, spin coating, in situ synthesis, and the like. Certaincoating materials can be cross-linked to the surface via heating,radiation, and/or by chemical reactions. In preferred aspects, suitablecoating materials are coupled to substrate surfaces either covalently orthrough ionic or hydrophobic/hydrophilic interactions. In the case ofsilica based substrates, for example, silane chemistries areparticularly suited for covalently attaching coating materials tosurfaces, e.g., coupling groups, specific binding moieties, and thelike. Such chemistries are well known to those of ordinary skill in theart and can be practiced without undue experimentation.

Fabrication Process:

Fabrication of the subject array substrates can be performed accordingto the methods described as follows or other standard techniques ofIC-processing and/or MEMS micromachining. The standard techniques knownin the art include but are not limited to electron-beam lithography,photolithography, chemical vapor or physical vapor deposition, dry orwet etching, ion implantation, plasma etching, bonding, andelectroplating. Additional fabrication processes are detailed in theU.S. patent application Publication No. 2003/0174992, the content ofwhich is incorporated by reference in its entirety.

In a preferred embodiment, the present invention provides a negativetone fabrication process, which provides for the creation of opticalconfinements having more uniform and consistent dimensions thanconventional positive tone fabrication processes that can yield opticalconfinements of varying dimensions. A comparison of the two fabricationprocesses is shown in Table 1 below. TABLE 1 Positive and Negative ToneProcess Steps in Fabrication of Zero-Mode Waveguides Step # PositiveTone Process Negative Tone Process 1 Clean fused silica substrates inheated solution of hydrogen Same peroxide and ammonium hydroxide. 2Cascade rinse substrates in deionized water. Same 3 Clean substrates inoxygen plasma cleaner. Same 4 Coat substrates with metal film by eitherthermal Spin-coat substrates with electron- evaporation or sputtering.beam resist. 5 Spin-coat substrates with electron-beam resist over theBake casting solvent out of film. metal layer. 6 Bake casting solventout of film. Expose resist with electron beam lithography. 7 Exposeresist with electron beam lithography. Develop resist in chemical bathto reveal array of small pillars with large empty gaps in resist. 8Develop resist in chemical bath to reveal holes. Rinse developer awayand dry chips. 9 Rinse developer away and dry chips. Coat chips withmetal film by either thermal evaporation or sputtering. 10 Usereactive-ion etching to transfer resist pattern into metal Dissolvingunderlying negative film. resist using Microposit 1165 Stripper. 11Strip resist using oxygen plasma. Same

In a negative tone process, a negative resist is applied to thesubstrate. A resist is negative if it is rendered insoluble byapplication of some agent, wherein the case of photoresists or e-beamresists, the agent is optical energy or electron beam energy,respectively. Alternatively, a positive tone resist can be used with anegative pattern. A negative tone pattern is characterized by theapplication of the agent in all areas except the location of the opticalconfinement, e.g., zero-mode waveguide, contrasted with a positive toneimage in which the agent is confined only to the optical confinementarea. In either case, after development of the resist, resist remainsonly in the areas where the optical confinement is intended to lie. Itis useful in many cases to use means to achieve an undercut sidewallprofile of these remaining resist features. Many techniques exist in theart to obtain undercut sidewalls, for example, in electron beamlithography. For instance, when using negative tone resists, one methodis to apply to layers of electron beam resist to the surfacesequentially, the upper film having a higher sensitivity to the energydelivered to it by the electron beam. Because the beam has a tendency tospread, a larger area of the upper film will be rendered insoluble thanin the lower layer, resulting in an overhang beneath the upper layer asdesired.

After development and appropriate cleaning procedures known in the artsuch as a plasma cleaning procedure, the metal film comprising theoptical confinement can be applied by one of several methods, includingmetal evaporation, molecular beam epitaxy and others. In the case thatthe resist profile is undercut as discussed above, the metal that isdeposited in the regions still occupied by the resist will rest on topof the resist rather than resting on the device surface. The resistlayer is subsequently removed by any of several techniques includingsolvent dissolution either with or without ultrasonication or othermechanical agitation, reactive plasma etching, vaporization or others.The metal which rested on the resist features is removed as the resistis removed (“lifted off”), while the resist resting directly on thesubstrate remains to form the walls of the optical confinement.

The advantage of this process is that the size of the opticalconfinement is determined by the size of the resist feature, and doesnot rely on the fidelity of reactive ion etch pattern transfermechanisms, which can be highly variable for metal films, especiallyaluminum a desirable metal for these devices. The positive tone processis subject to the inherent variation in resist feature sizes plus thevariation due to pattern transfer, while the negative tone process issubject to the first variability but not the second. Metal thin filmtechniques suffer from much less lateral variation, and so the overallaccuracy is better. This method also does not rely on the availabilityof a suitable etch for the metal in question, allowing the applicationof the process to a much wider selection of metals than the positivetone process.

FIG. 6 is a schematic presentation of an illustrative negative toneprocess to make zero-mode waveguides. In this process, the substrate 11is first coated with a layer of negative resist 12. Optionally, thesubstrate can be coated with a second resist layer 13. Exposure of theresist to the same pattern electron beam lithography tool used in thepositive tone process, generates the opposite pattern as previouslyobserved, namely one of a periodic array of small pillars of remainingresist, and empty gaps between the pillars 15. The final zero-modewaveguide structures are created by coating this pattern with a thinmetal layer such as an aluminum layer 17, and then dissolving theunderlying negative resist pillars 18. Because this process is notdependent on the thickness of the alumina layer or the crystal structureor morphology of the metal film, it produces a far more consistentconfiguration, and provides much finer control over the critical featuresize. FIG. 8 depicts a scanning electron micrographs of ZMW structuresfabricated by positive tone resist (left panels) or negative tone resist(right panels). The grain structure of the polycrystalline film isvisible in the image as flecks, and the ZMWs as dark round structures.

A variant negative tone process is termed nanocasting. The steps ofnanocasting are similar except that the use of bi-layer resist isavoided. The process first involves depositing on the surface of asubstrate (in this case a single-layer resist would be used). Theelectron beam exposure and development follow, leaving a cylindricalfeature for each dot in the exposure pattern. For this process, it isdesirable to allow the metal deposition technique to apply material notjust on the top of the resist structure but also on the sidewalls of theresist feature. This process is inherently three dimensional, in that anegative replica of the exterior surface of the three-dimensional resistfeature is reproduced in the interior surface of the metal films thatforms the optical confinement walls. In this case, the undercut resistprofile and the various methods used to produce this are not necessary,as in the negative tone process, they are used specifically to preventcontact of the deposited film with the sides of the resist feature. Inthe nanocasting approach, the deposited film faithfully reproduces theexterior surface of the resist feature, so an undercut figure would onlybe used if a non-cylindrical confinement is desired.

In practicing nanocasting, caution is typically employed to removed themetal from above the nanocasting “master” (the resist feature), as theresist feature can in some instances be entirely buried and unavailablefor removal. This, however, can be remedied in a number of ways.

Where the deposition technique has a high degree of anisotropy in thedeposition (such as metal evaporation), the sidewalls will be very thinnear the top of the resist feature, which in some instances can be acylindrical pillar. This weak point can be subject to direct mechanicaldisruption allowing the removal of the metal above the resist featureand hence the ZMW location. An isotropic etch, either solution phase orplasma can be used to further thin the film until this weak pointseparates, achieving the same effect. If the metal deposition step has alow degree of anisotropy (such as sputtering or electroplating), thenthe resist material can be exposed through chemical mechanicalpolishing, or ion milling.

Simultaneous with or subsequent to the removal of the metal cap over theresist feature, the resist material is then removed by solventdissolution, or reactive ion etching. This completes the fabricationsteps, provided the appropriate pattern is applied and the otherparameters are correctly chosen.

Uses of the Subject Optical Confinements and Other Devices:

The subject devices including optical confinements and associatedoptical systems provide a effective means for analyzing molecules andmonitoring chemical reactions in real time. The subject device anddetection/monitoring methods may be used in a wide variety ofcircumstances including analysis of biochemical and biological reactionsfor diagnostic and research applications. In particularly preferredaspects, the present invention is applied in the elucidation of nucleicacid sequences for research applications, and particularly in sequencingindividual human genomes as part of preventive medicine, rapidhypothesis testing for genotype-phenotype associations, in vitro and insitu gene-expression profiling at all stages in the development of amulti-cellular organism, determining comprehensive mutation sets forindividual clones and profiling in various diseases or disease stages.Other applications include measuring enzyme kinetics, and identifyingspecific interactions between target molecules and candidate modulatorsof the target molecule. Further applications involve profiling cellreceptor diversity, identifying known and new pathogens, exploringdiversity towards agricultural, environmental and therapeutic goals.

In certain embodiments, the subject devices and methods allowhigh-throughput single-molecule analysis. Single-molecule analysisprovides several compelling advantages over conventional approaches tostudying biological events. First, the analysis provides information onindividual molecules whose properties are hidden in the statisticallyaveraged information that is recorded by ordinary ensemble measurementtechniques. In addition, because the analysis can be multiplexed, it isconducive to high-throughput implementation, requires smaller amounts ofreagent(s), and takes advantage of the high bandwidth of optical systemssuch as modern avalanche photodiodes for extremely rapid datacollection. Moreover, because single-molecule counting automaticallygenerates a degree of immunity to illumination and light collectionfluctuations, single-molecule analysis can provide greater accuracy inmeasuring quantities of material than bulk fluorescence orlight-scattering techniques. As such, single-molecule analysis greatlyimproves the efficiency and accuracy in genotying, gene expressionprofiling, DNA sequencing, nucleotide polymorphism detection, pathogendetection, protein expression profiling, and drug screening.

Single-Molecule Sequencing:

The subject devices, including various forms of optical confinements andthe associated optical systems, are particularly suited for multiplexedsingle-molecule sequencing. Accordingly, the present invention providesa method of simultaneously sequencing a plurality of target nucleicacids. The method generally involves (a) providing an array of opticalconfinements of the present invention; (b) mixing in the confinements aplurality of target nucleic acid molecules, primers complementary to thetarget nucleic acid molecules, polymerization enzymes, and more than onetype of nucleotides or nucleotide analogs to be incorporated into aplurality of nascent nucleotide strands each being complimentary to arespective target nucleus and molecules; (c) subjecting the mixture to apolymerization reaction under conditions suitable for formation of thenascent nucleotide strands by template-directed polymerization; (d)illuminating the waveguides with an incident light beam; and (e)identifying the nucleotides or the nucleotide analogs incorporated intoeach nascent nucleotide strand.

The subject sequencing methods can be used to determine the nucleic acidof any nucleic acid molecule, including double-stranded orsingle-stranded, linear or circular nucleic acids (e.g., circular DNA),single stranded DNA hairpins, DNA/RNA hybrids, RNA with a recognitionsite for binding of the polymerase, or RNA hairpins. The methods of thepresent invention are suitable for sequencing complex nucleic acidstructures, such as 5′ or 3′ non-translation sequences, tandem repeats,exons or introns, chromosomal segments, whole chromosomes or genomes.

In one aspect, the temporal order of base additions during thepolymerization reaction is identified on a single molecule of nucleicacid. Such identifying step takes place while the template-directedextension of primer or polymerization is taking place within the opticalconfinement. In a preferred embodiment, single-molecule sequencing isperformed in a homogenous assay that does not require transfer,separation, or washing away any reactant or by-product (e.g. fluorophorecleaved from a nucleotide) after each base addition event. In certainaspects of the homogenous assay, single-molecule sequencing is performedwithout adding reactants to the mixture prior to reading the next basesequence. In this assay, stepwise addition of nucleotides or removal ofby-products after each base addition event is not necessary, asdiffusion of reactants from a large volume of reagents above theconfinement will not interfere with the detection of incorporation.Sequence information is generated continuously as the polymerasecontinually incorporates the appropriate nucleotides or nucleotideanalogs into the nascent DNA strand. For a detailed discussion of suchsingle molecule sequencing, see, e.g., Published U.S. patent applicationNo. 2003/0044781, which is incorporated herein by reference in itsentirety for all purposes and M. J. Levene, J. Korlach, S. W. Turner, M.Foquet, H. G. Craighead, W. W. Webb, SCIENCE 299:682-686, January 2003Zero-Mode Waveguides for Single-Molecule Analysis at HighConcentrations. There is no loss of synchronization because singlemolecules are observed separately. This method also allows the use oftarget nucleic acid molecules taken directly from a biological sample,minimizing the need for cloning, subcloning, or amplification of thetarget nucleic acids before sequencing can take place.

In a preferred embodiment, a polymerase enzyme is provided anchoredwithin the effective observation volume within an optical confinement.Template dependent synthesis of a complementary strand is then carriedout while observing the volume, and using labeled nucleotide analogsthat are capable of being sequentially incorporated into the growingstrand without interruption, e.g., for deprotection, etc. In preferredaspects, nucleotide analogs bearing a label on a non-incorporatedphosphate group or derivative, e.g., the beta, gamma, delta, etc.phosphate of a nucleotide polyphosphate which is cleaved from the analogduring incorporation, are used in such methods. Such nucleotide analogsprovide an advantage of being sequentially incorporated into the growingnucleic acid strand, and having their labeling groups removed in theincorporation process so as to not provide increasing signal noiseduring synthesis that would result if such labels remained associatedwith the synthesized strand. In addition, because the incorporationevent provides for prolonged presence of the labeled analogs within theobservation volume (as compared to random diffusion of non-incorporatedanalogs into the observation volume), the signal associated withinincorporation is readily identifiable. In particularly preferredaspects, for single molecule nucleic acid sequencing applications, atemplate nucleic acid is used that provides for the redundant oriterative reading/synthesis of tandem repeats of a particular sequencesegment of interest. In particular, the systems of the inventiontypically provide for redundancy in numerous ways, to correct for anyerrors that may arise in template dependant synthesis by the polymeraseenzyme. For example, because the methods of the invention focus onsingle molecules, redundant processes are employed to assure thatmis-incorporation events by a polymerase are corrected for in dataanalysis.

In a first aspect, such redundancy is supplied by utilizing arrays ofmultiple different confinements that are being applied to a givensequence of interest, e.g., in a single well of a multi-well plate. Inaddition to this redundancy, the invention also provides for theiterative sequencing of a given sequence segment (or a copy thereof)multiple times within a single confinement. In a first preferred aspect,such iterative sequencing may be accomplished by providing the sequencesegment of interest in a circular template format, so that thepolymerase processes around the circular template (allowing theelucidation of the sequence of such template) multiple times. Methods ofcircularization of nucleic acid segments are known to those of ordinaryskill in the art, and are readily applied to template sequences inaccordance with the invention.

In another aspect, a similar result is accomplished by using atemplate-dependant circular template bearing the sequence segment ofinterest. In particular, such synthesis product will typically include,in a single linear strand, multiple copies of the circular template,again, providing for iterative sequencing of the sequence segment ofinterest. Further, redundancy is additionally accomplished bycircularizing this linear, multi-copy template and iterativelysequencing multiple copies, multiple times.

In another aspect, a similar result is obtained by performingconcatmerization of amplicons generated in a single-moleculeamplification strategy, several of which are known to those skilled inthe art. These strategies can employ dilution to the single moleculelevel, or isolation of molecules in small micelles in a two-phaseemulsion during amplification. The concatmerized strand is thensequenced as a single template, and redundant information is generatedfrom a single molecule in this fashion.

In yet another aspect, a similar result is obtained by using a longdouble stranded template with nicks and/or gaps at multiple locationsalong it. The molecule can then be caused to initiate single moleculesequencing at several locations along the strand, each locationcomprising a confinement that independently sequences the strand.Because the several confinements are acting on the same strand, theresult is that the same template is sequenced several times providingredundant information from a single molecule.

Exemplary Experimental Setup:

In practicing a sequencing method of the present invention, a reactionmixture comprising the target nucleic acid(s) of interest, primerscomplementary to the target nucleic acids, polymerization enzymes, andmore than one type of nucleotides or nucleotide analogs, is applied toan array of optical confinements. Preferably, each optical confinementreceives only one target nucleic acid molecule that is to be sequenced.This can be achieved by diluting a minute amount of target nucleic acidsin a large volume of solution containing the rest of the reactantsrequired for the sequencing process. Alternatively, a non-cylindricalwaveguide whose the opening is narrower in lateral dimension than thebase, can be used to restrict the entry of multiple target nucleicacids.

Immobilization of the Target Nucleic Acid or the Polymerase to anOptical Confinement:

The target nucleic acid can be immobilized to the inner surface of theoptical confinement by a number of ways. For example, the target nucleicacid can be immobilized onto an optical confinement by attaching (1) aprimer or (2) a single-stranded target nucleic acid or (3)double-stranded or partially double-stranded target nucleic acidmolecule. Thereafter, either (1) the target nucleic acid molecule ishybridized to the attached oligonucleotide primer, (2) anoligonucleotide primer is hybridized to the immobilized target nucleicacid molecule to form a primed target nucleic acid molecule complex, or(3) a recognition site for the polymerase is created on thedouble-stranded or partially double-stranded target nucleic acid (e.g.,through interaction with accessory proteins, such as a primase). Anucleic acid polymerizing enzyme on the primed target nucleic acidmolecule complex is provided in a position suitable to move along thetarget nucleic acid molecule and extend the oligonucleotide primer atthe site of polymerization.

In preferred aspects, as described previously, the polymerization enzymeis first attached to a surface of the subject optical confinement withinthe effective observation volume of the confinement, and in a positionsuitable for the target nucleic acid molecule complex to move relativeto the polymerization enzyme.

One skilled in the art will appreciate that there are many ways ofimmobilizing nucleic acids and enzymes onto an optical confinement,whether covalently or noncovalently, via a linker moiety, or tetheringthem to an immobilized moiety. These methods are well known in the fieldof solid phase synthesis and micro-arrays (Beier et al., Nucleic AcidsRes. 27:1970-1-977 (1999). Non-limiting exemplary binding moieties forattaching either nucleic acids or polymerases to a solid support includestreptavidin or avidin/biotin linkages, carbamate linkages, esterlinkages, amide, thiolester, (N)-functionalized thiourea, functionalizedmaleimide, amino, disulfide, amide, hydrazone linkages, and amongothers. Antibodies that specifically bind to the target nucleic acids orpolymerases can also be employed as the binding moieties. In addition, asilyl moiety can be attached to a nucleic acid directly to a substratesuch as glass using methods known in the art.

Where desired, the polymerases may be modified to contain one or moreepitopes such as Myc, HA (derived from influenza virus hemagglutinin),poly-histadines, and/or FLAG, for which specific antibodies areavailable commercially. In addition, the polymerases can be modified tocontain heterologous domains such as glutathione S-transferase (GST),maltose-binding protein (MBP), specific binding peptide regions (seee.g., U.S. Pat. Nos. 5,723,584, 5,874,239 and 5,932,433), or the Fcportion of an immunoglobulin. The respective binding agents for thesedomains, namely glutathione, maltose, and antibodies directed to the Fcportion of an immunoglobulin are available, and can be used to coat thesurface of an optical confinement of the present invention.

The binding moieties or agents of either the polymerases or nucleicacids they immobilize can be applied to the support by conventionalchemical techniques which are well known in the art. In general, theseprocedures can involve standard chemical surface modifications of asupport, incubation of the support at different temperature levels indifferent media comprising the binding moieties or agents, and possiblesubsequent steps of washing and cleaning.

Reaction Mixture: Labeled Nucleotides, Polymerases, and Primers:

The various types of nucleotides utilized in accordance with thesingle-molecule sequencing method are conjugated with detectable labelsso that a photon detector can detect and distinguish their presencewithin the subject optical confinements. Preferred labels areluminescent labels, and especially fluorescent or chromogenic labels.

A variety of functional groups used as detectable labels in nucleotideshas been developed in the art. Table 1 lists numerous examples of suchfunctional groups. Additional examples are described in U.S. Pat. No.6,399,335, published U.S. patent application No. 2003/0124576, and TheHandbook—‘A Guide to Fluorescent Probes and Labeling Technologies, TenthEdition’ (2005) (available from Invitrogen, Inc.,/Molecular Probes), allof which are incorporated herein by reference. TABLE 1 Exemplarydetectable label functional groups 4-aminophenol 6-aminonaphthol4-nitrophenol 6-nitronaphthol 4-methylphenol 6-chloronaphthol4-methoxyphenol 6-bromonaphthol 4-chlorophenol 6-iodonaphthol4-bromophenol 4,4′-dihydroxybiphenyl 4-iodophenol 8-hydroxyquinoline4-nitronaphthol 3-hydroxypyridine 4-aminonaphthol umbelliferone4-methylnaphthol Resorufin 4-methoxynaphthol 8-hydroxypyrene4-chloronaphthol 9-hydroxyanthracene 4-bromonaphthol6-nitro9-hydroxyanthracene 4-iodonaphthol 3-hydroxyflavone6-methylnaphthol fluorescein 6-methoxynaphthol 3-hydroxybenzoflavone

Using these or other suitable functional groups known in the art, a vastdiversity of fluorophores suitable for the present sequencing method canbeen generated. They include but are not limited to4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalinide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonc acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylanino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron .RTM. Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives. Additional fluorophores applicable forthe subject sequencing methods are disclosed in U.S. Pat. No. 5,866,366and WO 01/16375, both of which are incorporated herein by reference.

The labels can be attached to the phosphate backbone, on the base, onthe ribose unit, or a combination thereof. Preferred labels are thosethat do not substantially impede the continuous addition of nucleotidesin a sequencing reaction. Such labels include those linked to the alphaphosphate, the beta phosphate, the terminal phosphate, or the delta ormore distal phosphates in tetra, penta or hexa phosphate nucleotides, orthe base unit of a nucleotide.

Nucleotides comprising labeled terminal phosphates (e.g., the gammaphosphate as in dNTP), are particularly preferred because no additionalmeans is required to remove the label in the sequencing procedure.During the process of nucleic acid polymerization, the bond cleavage inthe nucleotide occurs between the alpha and the beta phosphate, causingthe beta and terminal phosphate (e.g., the gamma phosphate as in dNTP)to be released from the site of polymerization. As such, the labelattached to the terminal phosphate is separated from the nascent strandonce the nucleotide is being incorporated. In general,terminal-phosphate-linked nucleotides may comprise three or morephosphates, typically about three to about six phosphates, preferablyabout three to about five phosphates. Table 1 lists numerous examples ofnucleotides with labeled terminal phosphates. Many otherterminal-phosphate-linked nucleotides have been developed and aredetailed in U.S. patent application No. 2003/0124576, which isincorporated herein by reference in its entirety. TABLE 2Adenosine-5′-(γ-4-nitrophenyl)triphosphateGuanosine-5′-(γ-4-nitrophenyl)triphosphateCytosine-5′-(γ-4-nitrophenyl)triphosphateThymidine-5′-(γ-4-nitrophenyl)triphosphateUracil-5′-(γ-4-nitrophenyl)triphosphate3′-azido-3′-deoxythymidine-5′-(γ-4-nitrophenyl)triphosphate3′-azido-2′,3′-dideoxythymidine-5′-(γ-4-nitrophenyl)triphosphate2′,3′-didehydro-2′,3′-dideoxythymidine-5′-(γ- 4-nitrophenyl)triphosphateAdenosine-5′-(γ-4-aminophenyl)triphosphateAdenosine-5′-(γ-4-methylphenyl)triphosphateAdenosine-5′-(γ-4-methoxyphenyl)triphosphateAdenosine-5′-(γ-4-chlorophenyl)triphosphateAdenosine-5′-(γ-4-bromophenyl)triphosphateAdenosine-5′-(γ-4-iodophenyl)triphosphateAdenosine-5′-(γ-4-nitronaphthyl)triphosphateAdenosine-5′-(γ-4-aminonaphthyl)triphosphateAdenosine-5′-(γ-4-methylnaphthyl)triphosphateAdenosine-5′-(γ-4-methoxynaphthyl)triphosphateAdenosine-5′-(γ-4-chloronaphthyl)triphosphateAdenosine-5′-(γ-4-bromonaphthyl)triphosphateAdenosine-5′-(γ-4-iodonaphthyl)triphosphateAdenosine-5′-(γ-6-methylnaphthyl)triphosphateAdenosine-5′-(γ-6-methoxynaphthyl)triphosphateAdenosine-5′-(γ-6-aminonaphthyl)triphosphateAdenosine-5′-(γ-6-nitronaphthyl triphosphateAdenosine-5′-(γ-6-chloronaphthyl)triphosphateAdenosine-5′-(γ-6-bromonaphthyl)triphosphateAdenosine-5′-(γ-6-iodonaphthyl)triphosphateAdenosine-5′-(γ-4′-hydroxybiphenyl)triphosphateAdenosine-5′-(γ-8-quinolyl)triphosphateAdenosine-5′-(γ-3-pyridyl)triphosphateAdenosine-5′-(γ-umbelliferone)triphosphateAdenosine-5′-(γ-resorufin)triphosphateAdenosine-5′-(γ-pyrene)triphosphateAdenosine-5′-(γ-anthracene)triphosphateAdenosine-5′-(γ-6-nitroanthracene)triphosphateAdenosine-5′-(γ-flavonyl)triphosphateAdenosine-5′-(γ-fluorescein)triphosphateAdenosine-5′-(γ-benzoflavone)triphosphateAdenosine-5′-(γ-(4-nitrophenyl)-γ′-(4-aminophenyl)triphosphateAdenosine-5′-(γ-(4-nitrophenyl)-γ′-(4-nitronaphthyl)triphosphate

Nucleotides comprising modified phosphate backbones can also be used.For example, the modified component can be a phosphordiamidate,methylphosphonate, alkyl phosphotriester, formacetal,phosphorodithioate, phosphothioate, phosphoramidothioate,phosphoramidate, or an analog thereof.

In some embodiments, the nucleotides or nucleotide analogs used in thepresent invention are reversible extension terminators comprisingreversible blocking groups. In some embodiments, the blocking group on areversible extension terminator is linked to a detectable label. Inother embodiments, the blocking group and the detectable label arelocated on different positions of a nucleotide. In yet otherembodiments, the blocking group is also a label.

An illustrative reversible extension terminator comprises a labeledribose unit at the 3′ end. Each label on the ribose unit, typically actsas a reversible blocking group that must be removed before the nextnucleotide addition event can take place during a polymerizationreaction. Preferred 3′-ribose labels comprise photo-removable functionalgroups that can be deprotected upon exposure to a light beam at asuitable wavelength.

In another example, the reversible blocking group is located at the 2′or the 4′ position of the ribose unit of a nucleotide. In yet anotherembodiment, the reversible blocking group is linked to or conjugated tothe base (adenine, thymine, cytosine, guanine, or uracil) a nucleotide.Non-limiting examples of reversible blocking groups, and especiallyphotocleavable blocking groups include but are not limited to thosemolecules depicted in FIGS. 14 and 15 and those described in theco-pending application Ser. No. 60/649,009, which is incorporated hereinby reference in its entirety.

The wavelength used to cleave the photocleavable blocking groups willdepend on the choice of the blocking group. The wavelength may rangefrom about 320 nm to about 800 nm. In some embodiment, the wavelengthfor cleaving the blocking group is about the same as the wavelength usedto detect the label. In other embodiments, the wavelength for cleavingthe blocking group is different from the wavelength used to detect thelabel.

In some embodiments, it is advantageous to use a mixture of labelednucleotides that is substantially free of unlabeled nucleotides. Suchcomposition and the uses thereof for sequencing are detailed inco-pending application Ser. No. 60/651,846, which is incorporatedherein. Briefly, the composition is prepared by treating a mixturecomprising labeled and unlabeled nucleotides or nucleotide analogs withan agent that specifically modifies unlabeled or incorrectly labelednucleotides or nucleotide analogs to reduce their ability to be used ina hybridization or sequencing assay. Preferably, the agent usedspecifically modifies unlabeled or incorrectly labeled nucleotidesanalogs to render them incapable of being used in a hybridization orsequencing assay. For example, the nucleotides can be modified so thatthey no longer contain structures generally needed for the Watson Crickbase pairing in a hybridization or template-directed sequencing assay.In some embodiments, for example, base units of the nucleotides aremodified. In some embodiments, phosphate groups, preferably terminalphosphate groups, of the nucleotides or nucleotide analogs are modifiedto yield molecules that are incorporated to a lesser extent into anascent nucleic acid strand during a template-directed polymerizationreaction. In more preferred embodiments, the terminal phosphate groupsof a nucleotide or nucleotide analogs are modified to yield moleculesthat cannot or that substantially cannot be incorporated into a nascentnucleic acid strand during a template-directed polymerization reaction.

The agents can comprise one or more enzymes. A variety of enzymes knownin the art are suitable for modifying the nucleotides or nucleotideanalogs, e.g. by cleaving or altering the configuration of the sugar,base, or phosphates, so as to disrupt the specific Watson Crick basepairing. Exemplary agents include but are not limited to guanine oradenine P-ribosyl transferase, purine nucleoside phosphorylase, AMPnuleosidase, nucleoside deoxyribosyl transferase for purines, andorotate P-ribosyl transferase, thymidine phosphorylase, thymidine oruridine nucleosidase, uridine phosphorylase, pyrimidine nucleosidephosphorylase nucleoside deoxyribosyl transferase.

Enzymes applicable for modifying the terminal phosphate groups ofnucleotides or nucleotide analogs include a wide array of phosphatases.An example of such enzyme is Shrimp Alkaline Phosphatase (SAP) that canremove the gamma and beta phosphates from a deoxynucleoside triphosphate(dNTP). The enzyme can convert specifically unlabeled dNTP into anucleoside monophosphate dNMP which is generally incapable of beingutilized by a polymerase enzyme in a template-directed sequencingreaction. It has been shown, that this phosphatase selectively modifynucleotides that are not labeled, e.g. at the terminal phosphate.Therefore, in a mixture of terminal phosphate-labeled and unlabelednucleotides, the SAP will preferentially act on unlabeled nucleotides,leaving a larger proportion of labeled nucleotides available forincorporation in a sequencing reaction.

Other suitable phosphatases that can be used include but are not limitedto calf intestinal alkaline phosphatases, and/or phosphatases of othermammals, crustaceans, and other animals. Examples of phosphatases thatmay be useful practicing the present invention can be found in U.S.20040203097, U.S. 20040157306, U.S. 20040132155; and U.S. 20040110180.

Any other naturally occurring or synthetic phosphatases or phosphatasesmade by recombinant DNA technology can also be used so long as theyspecifically or preferentially convert unlabeled nucleotides or analogs(as compared to labeled nucleotides), to molecules that aresubstantially incapable of being utilized by a polymerization enzyme.Directed molecular evolution can also be used to enhance and extend theactivity of related enzymes to yield the desired property describedabove. A wide variety of mutagenesis techniques, both in silicon and insitu, are available in the art. An example of a mutagenesis or screeningassay for generating such enzymes can involve a first test forabrogation of polymerization in the system with unlabeled nucleotides,and a second screen checking for the retention of polymerizationactivity in the presence of labeled nucleotides. Both of these screenscan be performed in the context of a highly multiplexed parallel assay.Enzymes showing some beneficial specificity can be retained, mutated bysome method, and then re-screened. Methods such as these have been shownto produce many orders of magnitude improvement in specificity andperformance.

Enzymes capable of selectively or preferentially modifying a subset ofunlabeled nucleotides can also be employed. For example, creatine kinaseenzyme is specific for the removal of a phosphate from adenosidetriphosphate, and will not act on other bases. Other enzymes thatselectively or preferentially act on one or more types of unlabelednucleotides can also be used.

The nucleotide modifying enzymes described above can be used topre-treat the nucleotides or nucleotide analogs, or can be used in thehybridization and/or sequencing reaction mixture, e.g., along with otherhybridization or sequencing reagents.

The reaction conditions under which the modification of the nucleotidestakes place will vary depending on the choice of the modifying enzymes.In one aspect, the conditions may be set within the followingparameters: pH is between 4.0 and 12.0, more preferably between pH 6.0and 10.0, more preferably between 7.0 and 9.0, more preferably less than8, more preferably between 7 and 8, and most preferably pH 7.5 and 8.5,preferably controlled by a buffer. The buffer can be Tris-basedpreferably at pH 7.5 to pH 8.5. Other buffers may be used such as, butnot limited to: organic buffers such as MOPS, HEPES, TRICINE, etc., orinorganic buffers such as phosphate or acetate. Buffers or other agentsmay be added to control the pH of the solution thereby increasing thestability of the enzymes. Where desired, reducing agent such as but notlimited to dithiotreitol (DTT) or 2-mercaptoethanol may be added tolimit enzyme oxidation that might adversely affect stability of theenzymes. The choice of specific reaction conditions including variousbuffers and pH conditions is within the skill of practitioners in thefield, and hence is not further detailed herein.

Upon completion of the pre-treatment, the enzymes can beheat-inactivated by raising the reaction temperature to at least about65° C., preferably between about 65° C. to about 80° C. Alternatively,the enzymes can be depleted from the reaction mixture by, e.g.,centrifugation through a filter (e.g., Millipore) that has a molecularweight cutoff smaller than the size of the enzyme.

After the treatment, the mixture generally comprises less than about30%, preferably less than about 20%, more preferably less than about10%, more preferably less than about 5%, more preferably less than about1%, more preferably less than about 0.5%, or more preferably less thanabout 0.1%, and even more preferably less than 0.01% of unlabelednucleotides or unlabeled nucleotide analogs. This enriched mixture oflabeled nucleotides or nucleotide analogs is particularly useful forhigh-resolution detection of the labeled nucleotides in asingle-molecule sequence reaction.

Importantly, the result of the foregoing treatment is a process forsynthesis of nucleic acids, preferably for elucidating a templatesequence using substantially only nucleotides, e.g., substantiallycomplete replacement of native nucleotides with nucleotide analogs, andparticularly labeled analogs. Such template dependant synthesis in thepresence of substantially only nucleotide analogs, and particularlylabeled analogs, also referred to as substantially complete replacement,in sequencing operations is considerably different from previouslydescribed sequencing methods where a single nucleotide is substitutedwith a labeled chain terminating nucleotide among the remaining threenatural nucleotides, or where a polymerase template complex areinterrogated with only one analog at a time to determine whether suchanalog is incorporated.

Another type of suitable nucleotides for the subject sequencing methodsallows detection via fluorescence resonance energy transfer (FRET). InFRET, an excited fluorophore (the donor) transfers its excited stateenergy to a light absorbing molecule (the acceptor) in adistance-dependent manner. The limitation on the distance over which theenergy can travel allows one to discern the interactions between labeledmolecules and entities in close proximity. Nucleotides of this type cancomprise a donor fluorophore attached to the base, ribose or preferablythe phosphate backbone (e.g., attached to the terminal phosphate), andan acceptor fluorophore attached to the base, ribose or the phosphatebackbone where the donor is not attached. In a preferred embodiment, thedonor fluorophore is attached to the terminal phosphate, and an acceptorfluorophore is linked to the base or the ribose unit of the nucleotide.Upon incorporation of this type of nucleotide into the nascent strand, afluorescent signal can be detected which can be caused by the release ofpoly-phosphate that is no longer quenched. By determining the order ofthe fluorescent poly-phosphate that is released upon incorporating acomplementary nucleotide during the polymerization event, one can deducethe base sequence of the target nucleic acid. Additional examples ofthis type of nucleotides is disclosed in U.S. application No.20030194740, which is incorporated herein by reference.

In another embodiment, the donor fluorophore can be present in anucleotide, and the acceptor is located in the polymerase, or viceversa. Where desired, the fluorophore in the polymerase can be providedby a green fluorescent protein (GFP) or a mutant thereof that has adifferent emission and/or absorption spectrum relative to the wildtypegreen fluorescent protein. For example, the GFP mutant H9-40 (Tsien etal., Ann. Rev. Biochem. 67: 509 (1998)) which is excited at 399 nm andemits at 511 nm, may serve as a donor fluorophore for use with BODIPY,fluorescein, rhodamine green and Oregon green. In addition,tetramethylrhodamine, Lissamine™, Texas Read and napthofluorescein canbe used as acceptor fluorophores with this GFP mutant.

Other representative donors and acceptors capable of fluorescence energytransfer include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonap-hthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′;6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,-2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)amin-ofluorescein (DTAF),2′,7′-dimethoxy4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron.™. Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phthalo cyanine; and naphthalo cyanine.

In alternative configurations, both donor and acceptor fluorophores maybe present upon each nucleotide analog, where the donor provides asubstantially uniform excitation spectrum, but donates energy to anacceptor that provides an emission spectrum that is different for eachtype of analog, e.g., A, T, G, or C. Such configurations provide anability to utilize a single excitation source for multiple differentemission profiles, reducing energy input requirements for the systemsutilized.

In addition, xanthene dyes, including fluoresceins and rhodamine dyescan be used as donor and acceptor pairs. Many of these dyes containmodified substituents on their phenyl moieties which can be used as thesite for bonding to the terminal phosphate or the base of a nucleotide.Where desired, acceptors acting as quenchers capable of quenching a widerange of wavelengths of fluorescence can be used. Representativeexamples of such quenchers include4-(4′-dimethylaminophenylaz-o)-benzoic acid (DABCYL), dinitrophenyl(DNP) and trinitrophenyl (TNP).

The polymerization enzymes suitable for the present invention can be anynucleic acid polymerases that are capable of catalyzingtemplate-directed polymerization with reasonable synthesis fidelity. Thepolymerases can be DNA polymerases or RNA polymerases, a thermostablepolymerase or a thermally degradable polymerase wildtype or modified.Non-limiting examples for suitable thermostable polymerases includepolymerases from Thermus aquaticus, Thermus caldophilus, Thermusfiliformis, Bacillus caldotenax, Bacillus stearothermophus, Thermusthermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcuslitoralis, and Thermotoga maritima. Useful thermodegradable polymersasesinclude E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, T4 DNA polymerase, T7 DNA polymerase.

Additional examples of polymerization enzymes that can be used todetermine the sequence of nucleic acid molecules include E. coli T7, T3,SP6 RNA polymerases and AMV, M-MLV and HIV reverse transcriptases. Thepolymerase can be bound to the primed target nucleic acid sequence at aprimed single-stranded nucleic acid, an origin of replication, a nick orgap in a double-stranded nucleic acid, a secondary structure in asingle-stranded nucleic acid, a binding site created by an accessoryprotein, or a primed single-stranded nucleic acid.

In one preferred embodiment, the polymerization enzymes exhibit enhancedefficiency as compared to the wildtype enzymes for incorporatingunconventional or modified nucleotides, e.g., nucleotides linked withfluorophores. Recombinant DNA techniques can be used to modify thewildtype enzymes. Such techniques typically involve the construction ofan expression vector or a library of expression vector, a culture oftransformed host cells under such condition such that expression willoccur. Selection of the polymerases that are capable of incorporatingunconventional or modified nucleotides can be carried out using anyconventional sequencing methods as well as the sequencing methodsdisclosed herein.

In another preferred embodiment, sequencing is carried out withpolymerases exhibiting a high degree of processivity, i.e., the abilityto synthesize long stretches of nucleic acid by maintaining a stablenucleic acid/enzyme complex. A processive polymerase can typicallysynthesize a nascent strand over about 10 kilo bases. With the aid ofaccessory enzymes (e.g., helicases/primases), some processivepolymerases can synthesize even over 50 kilobases. For instance, it hasbeen shown that T7 DNA polymerase complexed with helicase/primase cansynthesize several 100 kilobases of nucleotides while maintaining astable complex with the target nucleic acid (Kelman et al.,“Processivity of DNA Polymerases: Two Mechanisms, One Goal” Structure 6:121-125 (1998)).

In another preferred embodiment, sequencing is performed withpolymerases capable of rolling circle replication, i.e., capable ofreplicating circular DNA templates including but not limited to plasmidsand bacteriophage DNA. A preferred rolling circle polymerase exhibitsstrand-displacement activity, and preferably has reduced or essentiallyno 5′ to 3′ exonuclease activity. Strand displacement results in thesynthesis of tandem copies of a circular DNA template, thus allowingre-sequencing the same DNA template more than once. Re-sequencing hesame DNA template greatly enhances the chances to detect any errors madeby the polymerase, because the same errors unlikely would be repeated bythe polymerase and the same error certainly would not be exponentiallyamplified as in a polymerase chain reaction.

Non-limiting examples of rolling circle polymerases suitable for thepresent invention include but are not limited to T5 DNA polymerase(Chatterjee et al., Gene 97:13-19 (1991)), and T4 DNA polymeraseholoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149-157 (1995)), phageM2 DNA polymerase (Matsumoto et al., Gene 84:247 (1989)), phage PRD1 DNApolymerase (Jung et al., Proc. Natl. Aced. Sci. USA 84:8287 (1987), andZhu and Ito, Biochim. Biophys. Acta. 1219:267-276 (1994)), Klenowfragment of DNA polymerase I (Jacobsen et al., Eur. J. Biochem.45:623-627 (1974)).

A preferred class of rolling circle polymerases utilizes protein primingas a way of initiating replication. Exemplary polymerases of this classare modified and unmodified DNA polymerase, chosen or derived from thephages Φ29, PRD1, Cp-1, Cp-5, Cp-7, Φ15, Φ1, Φ21, Φ25, BS 32 L17, PZE,PZA, Nf, M2Y (or M2), PR4, PR5, R722, B 103, SF5, GA-1, and relatedmembers of the Podoviridae family. Specifically, the wildtypebacteriophage Φ29 genome consists of a linear double-stranded DNA(dsDNA) of 19,285 base pairs, having a terminal protein (TP) covalentlylinked to each 5′ end. To initiate replication, a histone-like viralprotein forms a nucleoprotein complex with the origins of replicationthat likely contributes to the unwinding of the double helix at both DNAends (Serrano et al., The EMBO Journal 16(9): 2519-2527 (1997)). The DNApolymerase catalyses the addition of the first dAMP to the hydroxylgroup provided by the TP. This protein-primed event occurs opposite tothe second 3′ nucleotide of the template, and the initiation product(TP-dAMP) slides back one position in the DNA to recover the terminalnucleotide After initiation, the same DNA polymerase replicates one ofthe DNA strands while displacing the other. The high processivity andstrand displacement ability of Φ29 DNA polymerase makes it possible tocomplete replication of the Φ29 TP-containing genome (TP-DNA) in theabsence of any helicase or accessory processivity factors (reviewed bySerrano et al., The EMBO Journal 16(9): 2519-2527 (1997)).

Modified Φ29 DNA polymerases having reduced 5′ to 3′ exonucleaseactivity have also been described (U.S. Pat. Nos. 5,198,543 and5,001,050, both being incorporated herein). These polymerases areparticularly desirable for sequencing as the 5′ to 3′ exonucleases, ifpresent excessively, may degrade the nascent strand being synthesized.

Strand displacement can be enhanced through the use of a variety ofaccessory proteins. They include but are not limited to helicases(Siegel et al., J. BioL Chem. 267:13629-13635 (1992)), herpes simplexviral protein ICP8 (Skaliter and Lehman, Proc. Natl, Acad. Sci. USA91(22):10665-10669 (1994)), single-stranded DNA binding proteins (Riglerand Romano, J. Biol. Chem. 270:8910-8919 (1995)), adenovirus DNA-bindingprotein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164(1994)), and BMRF1 polymerase accessory subunit (Tsurumi et al., J.Virology 67(12):7648-7653 (1993)).

In a preferred embodiment, the sequence reaction involves a singlecomplex of strand-displacement polymerization enzyme and a circulartarget DNA, which is immobilized to an optical confinement. Upon mixingthe labeled nucleotides or nucleotide analogs and the primers, thestrand-displacement polymerization enzyme directs the synthesis of anascent strand and a time sequence of incorporating the various types oflabeled nucleotides or nucleotide analogs into the nascent strand isregistered. Where desired, the strand-displacement polymerase is allowedto synthesize multiple tandem repeats of the target DNA, and thuseffecting re-sequencing the same circular DNA target multiple times. Itis preferably to register the time sequence of the nucleotides ornucleotide analogs incorporated into at least two tandem repeats of thetarget DNA molecule, more preferably at least about three to about tenor about three to about one hundred tandem repeats, and preferably nomore than about one million repeats. This multiple rounds of orredundant sequencing can take place under an isothermal condition and/orat ambient temperature.

Using the subject method, sequencing can be carried out at the speed ofat least 1 base per second, preferably at least 10 bases per second,more preferably at least 100 bases per second. It has been reported thatpolymerases can polymerize 1,000 bases per second in vivo and 750 basesper second in vitro (see, e.g. Kelman et al., “Processivity of DNAPolymerases: Two Mechanisms, One Goal,” Structure 6: 121-125 (1998);Carter et al., “The Role of Exonuclease and Beta Protein of Phage Lambdain Genetic Recombination. II. Substrate Specificity and the Mode ofAction of Lambda Exonuclease,” J. Biol. Chem. 246: 2502-2512 (1971);Tabor et al., “Escherichia coli Thioredoxin Confers Processivity on theDNA Polymerase Activity of the Gene 5 Protein of Bacteriophage T7,” J.Biol. Chem. 262: 16212-16223 (1987); and Kovall et al., “ToroidalStructure of Lambda-Exonuclease” Science 277: 1824-1827 (1997), whichare hereby incorporated by reference).

Reaction Conditions:

The sequencing procedures of the present invention are performed underany conditions such that template-directed polymerization can take placeusing a polymerization enzyme. In one aspect, the substrates of thepolymerization enzyme, namely the various types of nucleotides presentin the sequence reaction, are adjusted to a physiologically relevantconcentration. For example, the nucleotides used in the sequencingreaction are present at a concentration about Michaelis constant of thepolymerization enzyme. Such concentration typically ranges from about 1micromolar to about 50 micromolar or about 100 micromolar.

The sequencing procedures can also be accomplished using less than fourlabels employed. With three labels, the sequence can be deduced fromsequencing a nucleic acid strand (1) if the fourth base can be detectedas a constant dark time delay between the signals of the other labels,or (2) unequivocally by sequencing both nucleic acid strands, because inthis case one obtains a positive fluorescence signal from each basepair. Another possible scheme that utilizes two labels is to have onebase labeled with one fluorophore and the other three bases with anotherfluorophore. In this case, the other three bases do not give a sequence,but merely a number of bases that occur between the particular basebeing identified by the other fluorophore. By cycling this identifyingfluorophore through the different bases in different sequencingreactions, the entire sequence can be deduced from sequential sequencingruns. Extending this scheme of utilizing two labels only, it is evenpossible to obtain the full sequence by employing only two labelledbases per sequencing run.

The sequencing procedures can be performed under an isothermalcondition, at ambient temperature, or under thermal cycling condition.The choice of buffers, pH and the like is within the skill ofpractitioners in the art, and hence is not detailed herein.

Detection:

The subject sequencing method requires the imaging of individualmolecules confined in an optical confinement. The polymerase and/or thenucleotides are labeled with fluorophores that emit a distinguishableoptical signal when a particular type of nucleotide is incorporated intothe nascent strand. The sequence of the distinguishable signals isdetected as the nucleotides are sequentially added to the nascent strandwithin the optical confinement. In a preferred embodiment, suchdetection is performed without the need to transfer, separation orwashing away any reactant or by-product (e.g. fluorophore cleaved from anucleotide) after each nucleotide addition event. In one aspect of thispreferred embodiment, sequence detection is performed without addingreactants to the mixture prior to reading the next base sequencenucleotide to be incorporated.

Imaging individual molecules confined in the subject opticalconfinements is performed with the aid of an optical system. Such systemtypically comprises at least two elements, namely an excitation sourceand a photon detector. Numerous examples of these elements are describedabove.

In a preferred embodiment, the excitation source is a laser, preferablya polarized laser. The choice of laser light will depend on thefluorophores attached to the different type of nucleotides and/or thepolymerases. For most of the flurophorescent compounds, the requiredexcitation light is within the range of about 300 nm to about 700 nm.For proteinaceous fluorophores such as green-flurorescent protein andmutants thereof, the excitation wavelength may range from about 488 nmto about 404 nm. Those skilled in the art will know or will be able toascertain the appropriate excitation wavelength to excite a givenfluorophore by routine experimentation (see e.g., The Handbook—‘A Guideto Fluorescent Probes and Labeling Technologies, Tenth Edition’ (2005)(available from Invitrogen, Inc./Molecular Probes) previouslyincorporated herein by reference).

Another consideration in selecting an excitation source is the choicebetween one-photon and multiphoton excitation of fluorescence.Multiphoton excitation coupled with detection, also known as multiphotonmicropscopy (“MPM”), provides enhanced sensitivity and spatialresolution. MPM is a form of laser-scanning microscopy that useslocalized nonlinear excitation to excite fluorescence within a thinraster-scanned plane. In MPM, as in conventional laser-scanning confocalmicroscopy, a laser is focused and raster-scanned across the sample. Theimage consists of a matrix of fluorescence intensity measurements madeby digitizing the detector signal as the laser sweeps back and forthacross the sample. Two-photon excitation probabilities are extremelysmall, and focusing increases the local intensity at the focal point.Although two-photon excited fluorescence is usually the primary signalsource in MPM, three-photon or more excited fluorescence and second orthird-harmonic generation can also be used for imaging. See, e.g., areview of multiphoton micropscopy in Webb et al. Nature Biotechnology(2003) 21: (11) 1251-1409. A preferred MPM setup comprises MPM laserscanning microscopes and second-harmonic imaging, equipped withfemtosecond mode-locked titanium sapphire lasers operating atwavelengths from about 700 to 1,000 nm. Such setup can capture more thanabout 100 photons per pixel in most of the conventional imagingmultiphoton microscope.

The sequence of the distinguishable signals can also be detected byother optical systems comprising elements such as optical reader,high-efficiency photon detection system, photo multiplier tube, gatesensitive FET's, nano-tube FET's, photodiode (e.g. avalanche photodiodes (APD)), camera, charge couple device (CCD), electron-multiplyingcharge-coupled device (EMCCD), intensified charge coupled device (ICCD),and confocal microscope.

A preferred combination comprises wide field CCD or ICCD and intensifiedvideo imaging microscopes with digital image processing capability, aswell as Fluorescence Photobleaching Recovery (FPR) and FluorescenceCorrelation Spectroscopy (FCS) coupled with confocal multiphotoncapability and continuous data acquisition and control. Such set up mayfurther comprise modular instrument for quasi-elastic light scattering,laser DIC interferometry, correlation spectroscopy instrumentation,components of optical force microscopy, and Time Correlated SinglePhoton Counting.(TCSPC).

These optical systems may also comprise optical transmission elementssuch as diffraction gratings, arrayed waveguide gratings (AWG), opticfibers, optical switches, mirrors, lenses (including microlens andnanolens), collimators. Other examples include optical attenuators,polarization filters (e.g., dichroic filter), wavelength filters(low-pass, band-pass, or high-pass), wave-plates, and delay lines. Insome embodiments, the optical transmission element can be planarwaveguides in optical communication with the arrayed opticalconfinements.

These and other optical components known in the art can be combined andassembled in a variety of ways to effect detection of thedistinguishable signals emitted from the sequencing reaction. Preferreddevices allow parallel data collection using arrays having a largenumber of optical confinements, where simultaneous and independentsequencing of nucleic acids takes place. In one aspect, the preferredsystem can collect and process signals from more than 10⁴ opticalconfinements, more than 2×10⁴ optical confinements, or more than 10⁵optical confinements, or more than 2×10⁵ optical confinements, orpreferably more than 10⁶, or preferably more than 2×10⁶ opticalconfinements, and even more preferably more than 10⁷ or 2×10⁷ opticalconfinements. In another aspect, the preferred setup can monitor in realtime the simultaneous and independent sequencing of nucleic acids at aspeed of about 1 base per second, preferably at a speed of about 10bases per second, more preferably at a speed of about 100 bases persecond and even more preferably at 1,000 bases per second. As such, themassive parallelism coupled with the rapid sequencing reaction canprovide an overall sequencing output greater than 100,000 bases persecond. The overall output can be scaled up to at least 1 megabase persecond, preferably 10 or more megabases per second. Further by obtainingsuch date from multiple different sequence fragments e.g., in from twoor more different reaction volumes, one can obtain independentsequences, e.g. from contiguous fragments of genomic DNA, allowing thehigh rate of throughput that is directly applicable to genomicsequencing.

Other Single-Molecule Applications:

The subject optical confinements and arrays of optical confinements findutility in many other chemical and biological applications where singlemolecule analyses are desired. In general, the subject opticalconfinements are applicable for any single molecule analysis involvingany reagent that can be attached to the surface and for which substratescan be labeled, including, enzymes, nucleic acids, antibodies, antigens,and the like. Such applications include discerning interactionsinvolving biological molecules such as proteins, glycoproteins, nucleicacids, and lipids, as well as inorganic chemicals, or any combinationsthereof. The interactions may be between nucleic acid molecules, betweennucleic acid and protein, and between protein and small molecules.

Abnormalities in interactions involving biological molecules have longbeen acknowledged to account for a vast number of diseases including,numerous forms of cancer, vascular diseases, neuronal, and endocrinediseases. An abnormal interaction, in form of e.g., constitutiveactivation and premature inactivation of a signaling complex, are nowknown to lead to aberrant behavior of a disease cell. In the case ofcancer, abnormal interactions between two signaling transductionmolecules, such as growth factor receptors and their correspondingligands, may result in dysfunction of cellular processes, whichultimately lead to dysregulated growth, lack of anchorage inhibition,genomic instability and/or propensity for cell metastasis.

A specific interaction between biological or chemical moleculestypically involves a target molecule that is being investigated and aprobe suspected to be able to specifically interact with the target. Inpracticing the subject methods, the target and the probe are placedwithin an optical confinement. The target-probe complex can be aprotein-protein complex, a glycoprotein-protein complex (e.g., receptorand ligand complex), a protein-nucleic acid complex (e.g., transcriptionfactor and nucleic acid complex), a protein-lipid complex, and complexof inorganic or organic small molecules.

Preferably, each optical confinement contains only one target that isbeing investigated. This can be achieved by diluting a minute amount oftarget in a large volume of solution, such that deposition over an arrayof confinements results in a primary distribution, or a majority ofconfinements will have a single target molecule disposed there.Alternatively, a non-cylindrical waveguide, wherein the opening of thewaveguide core is narrower in lateral dimension than the base, can beused to restrict the entry of multiple target proteins while permittingthe entry of a number of smaller probes.

The target or probe can be immobilized onto the inner surface of theoptical confinement by any of the methods applicable for immobilizingand depositing the polymerases described in the section above. Suchmethods encompass the uses of covalent and noncovalent attachmentseffected by a variety of binding moieties. The choice of the bindingmoieties will depend on the nature of the target and/or the probe. Forexample, the binding moieties can be synthetically linked to the proteintarget or the probe, or made as a fusion motif or tag via a recombinantmeans. A preferred way to immobilize the target protein or theproteinaceous probe involves the use of the streptavidin oravidin/biotin binding pair, and any other binding moieties or agentsdescribed above.

The reaction conditions will depend on the particular interaction thatis under investigation. One may vary the reaction temperature, theduration of the reaction, the buffer strength, and the targetconcentration or the probe concentration. For example, one may vary theconcentration of the probe in order to measure its binding affinity tothe target protein. To determine the thermal stability of thetarget-probe complex, one may vary the reaction temperature. Stabilityof the target-probe complex can also be determined by varying the pH, orbuffer salt concentration. Where desired, the interaction can be studiedunder physiologically relevant temperature and buffer conditions. Aphysiologically relevant temperature ranges from approximately roomtemperature to approximately 37° C. A physiological buffer contains aphysiological concentration of salt at neutral pH ranging from about 6.5to about 7.8, and preferably from about 7.0 to about 7.5. Adjusting thereaction conditions to discern a particular interaction in vitro betweena given target and a probe is within the skill of artisans in the field,and hence is not detailed herein.

The target and/or the probe are generally labeled with detectable labelsso that a photon detector can detect a signal indicative of theirinteraction. Suitable labels encompass all of those labels disclosed inthe Single-Molecule Sequencing section. Preferred labels are luminescentlabels, and especially fluorescent or chromogenic labels.

In one embodiment, the target is labeled with a fluorophore whose signalis quenched upon interaction with the corresponding probe conjugatedwith an appropriate quencher. A variety of suitable fluorophore-quencherpairs is disclosed in the section above and hence is not detailedherein. A variation of this embodiment is to label the target and theprobe with donor and acceptor fluorphores (or vise versa) that emit adistinguishable signal when the two molecules bind to each other. A widerange of applicable donor and acceptor fluorophores is also describedabove. Those of skill in the art will appreciate the wide diversity ofdetectable labels and the combinations thereof to generate adistinguishable signal that is indicative of a specific interactionbetween biological molecules and/or chemical compounds.

The detection of the distinguishable signal indicative of a specificinteraction is performed with the aid of the optical systems describedherein. Any of the systems applicable for single-molecule sequencing isequally suited for detecting interactions between other biologicalmolecules and/or chemical compounds. A preferred system allows paralleldata collection using arrays having a large number of opticalconfinements, where simultaneous and independent target-probeinteractions can take place. In one aspect, the preferred system cancollect and process signals from more than 10⁴ optical confinements,more than 2×10⁴ optical confinements, more than 10⁵ opticalconfinements, more than 2×10⁵ optical confinements, preferably more than10⁶, or preferably more than 2×10⁶ optical confinements, and even morepreferably more than 10⁷ or 2×10⁷ optical confinements.

Of particular significance is the application of the aforementionedmethod in detecting the presence of a specific protein-proteininteraction. Such application generally employs a proteinaceous probeand a target protein placed in an optical confinement. In one aspect ofthis embodiment, the specific protein-protein interaction is between acell surface receptor and its corresponding ligand. Cell surfacereceptors are molecules anchored on or inserted into the cell plasmamembrane. They constitute a large family of proteins, glycoproteins,polysaccharides and lipids, which serve not only as structuralconstituents of the plasma membrane, but also as regulatory elementsgoverning a variety of biological functions. In another aspect, thespecific protein-protein interaction involves a cell surface receptorand an immunoliposome or an immunotoxin. In yet another aspect, thespecific protein-protein interaction may involve a cytosolic protein, anuclear protein, a chaperon protein, or proteins anchored on otherintracellular membranous structures. In yet another aspect, the specificprotein-protein interaction is between a target protein (e.g., anantigen) and an antibody specific for that antigen.

The specific interaction between an antigen and an antibody has beenexplored in the context of immunoassays. There exists a variety ofimmunoassays in the art, but none of which permits single-moleculedetection. For instance, the conventional radioimmunoassay detects theinteractions between a population of antigens and a population ofradioactively labeled antibodies on an immunoblot. Another conventionalimmunoassay termed ELISA (Enzyme Llinked Immunoradiometric Assay)utilizes an antigen-specific antibody and an enzyme-lined genericantibody that binds to the specific antibody. The specific interactionbetween the antigen and the antibody is visualized upon addition of thesubstrate to the linked enzyme. Such assay again is performed on animmunoblot providing an ensemble measurement of all interactionsdetected.

The subject optical confinement provides an effective tool forconducting a single-molecule immunoassay. Unlike the conventionalimmunoassays, the specific interaction between the antigen and theantibody can be resolved at the single-molecule level. While all of theoptical confinements embodied in the present invention are applicablefor conducting single-molecule immunoassays, a particularly desirablesystem comprises an array of optical confinements with a relatively highfill fraction ratio. For example, a preferred system comprises an arrayof waveguides having a fill fraction greater than 0.0001, morepreferably greater than about 0.001, more preferably greater than about0.01, and even more preferably greater than 0.1.

In practicing the subject immunoassays, the antibodies an be labeledwith a suitable label selected from radioactive labels, fluorescentlabels, chemiluminescent labels, enzyme tags, such as digoxigenin,β-galactosidase, urease, alkaline phosphatase or peroxidase,avidin/biotin complex, and any of the detectable labels disclosedherein.

The subject immunoassays can be performed to characterize biologicalentities, screen for antibody therapeutics, and determine the structuralconformations of a target antigen. For instance, immuoassays involvingantibodies that are specific for the biological entity or specific for aby-product produced by the biological entity have been routinely used toidentify the entity by forming an antibody-entity complex. Immunoassaysare also employed to screen for antibodies capable of activating ordown-regulating the biological activity of a target antigen oftherapeutic potential. Immunoassays are also useful for determiningstructural conformations by using anti-idotypic antibodies capable ofdifferentiating target proteins folded in different conformations.

Another important application of the aforementioned single-moleculeanalysis is to study enzyme kinetics, which may include determining theenzymatic turnover cycle, the dynamic behavior, folding and unfoldingintermediates, and binding affinities. The enzymes under investigationmay be immobilized within the optical confinements or present insolutions confined within the subject optical confinements.

All of the optical confinements embodied by the present invention can beemployed to study enzyme kinetics. The choice of a specific opticalconfinement will depend on the specific characteristic that is underinvestigation. For instance, an optical confinement comprising anon-cylindrical core having an opening on the upper surface that isnarrower than that of the base of the optical confinement is preferablefor measuring the association rate constant (on-rate) of an enzymaticreaction. This configuration significantly restricts the diffusion ofreactants or substrates, and hence increases the average residence timein the observation volume. On the other hand, an optical confinementcomprising a core with an opening that is wider in lateral dimensionthan the base imposes impose a stearic or entropic hinderence toentering the structure, hence is useful for measuring the accessibilityfor large enzymes or enzymatic complexes.

Uses of the Subject Optical Confinements in Ensemble Measurements:

While the optical confinements of the present invention are particularlyuseful in conducting single-molecule analyses, the subject confinementsare also suited for high throughput performance of ensemble bulkmeasurements. Accordingly, the present invention provides a method ofdetecting interactions among a plurality of molecules, comprising:placing said plurality of molecules in close proximity to an array ofzero-mode waveguides, wherein individual waveguides in said array areseparated by a distance sufficient to yield a detectable intensity ofdiffractive scattering at multiple diffracted orders upon illuminatingsaid array with an incident wavelength of light beam; illuminating saidarray of zero-mode waveguides with said incident wavelength; anddetecting a change in said intensity of diffractive scattering of saidincident wavelength at said multiple diffracted orders, therebydetecting said interactions among said plurality of molecules.

Arrays employed for this method typically comprises optical confinementsspaced far apart relative to the incident wavelength. Such spacing ofthe individual optical confinements far apart relative to theilluminating radiation (e.g., half of the wavelength of the illuminatingradiation) creates a larger effect on the diffractive scattering ofincident light at a given angle away from the angle of speculareflection. In one aspect of this embodiment, the arrays containindividual confinements separated by more than one wavelength of theincident radiation, usually more than 1.5 times the incident wavelength,but usually does not exceed 150 times the incident wavelength.

Arrays having the optical confinements spaced far apart relative to theincident wavelength also have desirable properties. While theangle-dependent scattering may raise the background signal that could bedisadvantageous for certain applications, it provides a meansparticularly suited for characterizing the size and shape of the opticalconfinements. It also readily permits ensemble bulk measurements ofmolecule interactions, involving especially unlabelled molecules. Arrayssuited for such applications generally contain individual confinementsseparated by more than one wavelength of the incident radiation, usuallymore than 1.5 times the incident wavelength, but usually not exceeding150 times the incident wavelength.

The ensemble bulk measurement is typically performed with the aid of theoptical systems described herein. Any of the setup applicable forsingle-molecule sequencing is equally suited for this analysis.

Further illustrations of the fabrication of the optical confinements ofthe present invention and then uses in sequencing are provided in theExample section below. The examples are provided as a guide to apractitioner of ordinary skill in the art, and are not meant to belimiting in any way.

EXAMPLES Example 1

The following provides an illustrative process of fabricating zero-modewaveguide. The parameters described herein are meant to be illustrativeand not intended to be limiting in any manner.

-   -   1. Substrates: Substrates are double polished, 60/40 scratch/dig        surface quality, Fused Silica wafers, cut to        100millimeters(±0.2 mm) diameter, and 175 micrometer(±25        micrometers) thick and a total thickness variation of less than        25 micrometers.    -   2. Clean: A mix of 5 parts deionized water, 1 part of (30% v/v        Hydrogen Peroxide in water), 1 part of (30% v/v Ammonium        Hydroxide in water) is heated to 75 degree Celsius on a        hotplate. The wafers are immersed in the mix using a Teflon        holder or other chemically resistant holder for a duration of 15        minutes.    -   3. Rinsing: The holder containing the wafers is removed from the        RCA clean bath and immersed in a bath of deionized water. The        wafers are left in this second bath for a 2 minutes period. The        holder still containing the wafers is removed from the bath, and        sprayed with deionized water to thoroughly finish the rinsing        process.    -   4. Drying: Within a minute of the final rinsing step, the wafers        are dried, while still in the holder, using a dry clean nitrogen        flow.    -   5. Oxygen Plasma: The wafers are then placed in a Glenn 1000p        plasma Asher, used in plasma etch mode (wafers on a powered        shelf, and under another powered shelf), with 140 mTorr pressure        and 400 Watts of forward power at 40 kHz frequency. The plasma        is maintained for 10 minutes. A flow of 18 sccm of molecular        oxygen is used.    -   6. Vapor Priming: The wafers are loaded within 3 minutes after        the Oxygen plasma in a Yield Engineering Systems vapor priming        oven where they are coated with a layer of HexaMethylDiSilazane        (HMDS) adhesion promoter.    -   7. Electron beam resist coating: The wafers are coated within 15        minutes after the Vapor Priming in a manual spinner unit using        NEB-31 electron beam resist (Sumitomo Chemical America). About 3        ml are dispensed on the wafer, which is then spun at 4500 rpm        for 60 seconds. Initial acceleration and deceleration are set to        3 seconds    -   8. Resist Bake: The wafers are baked on a CEE hotplate at a        temperature of 115 degree Celsius for 2 minutes. The plate is        equipped with a vacuum mechanism that allows good thermal        contact between the wafers and the hotplate surface.    -   9. Gold Evaporation: a layer of 10 nm of gold is then thermally        evaporated on the Wafers, on the side coated with the resist. A        pressure of less than 2 10e-06 Torr must be reached before the        evaporation. The evaporation is performed at a rate of        approximately 2.5 Angstrom per second and monitored using an        Inficon controller.    -   10. Electron beam exposure: a pattern consisting of Zero Mode        Waveguides is exposed on the wafers, using a high resolution        electron beam lithography tool such as a Leica VB6-HR system.        Zero mode waveguides are patterned as single exel features. At a        current of nominally 1 nanoAmpere, and a Variable Resolution        Unit of 1, and for an exel setting of 5 nanometers, doses can        range from 10000 microCoulombs per square centimeters to 300000        microCoulombs per square centimeters.    -   11. Post Exposure Bake: The wafers are then submitted to a 2        minute post exposure bake on a hotplate at 95 degree Celsius,        equally equipped with a vacuum mechanism.    -   12. Gold Etch: After removal from the electron beam system, the        10 nanometer gold layer is removed using gold etchant TFA at        room temperature (GE 8148, Transene Corporation), for 10        seconds. Wafers are held in a Teflon holder similar to the one        used in step 2.    -   13. Rinsing: The holder containing the wafers is removed from        the gold etchant bath and immerse in a bath of deionized water.        The wafers are left in this second bath for a 2 minutes period        or shorter with gentle manual agitation. The holder still        containing the wafers is removed from the bath, and sprayed with        deionized water to thoroughly finish the rinsing process.        Alternatively, the holder still containing the wafer is then        placed into a new container containing fresh deionized water.    -   14. Drying: Within a minute of the final rinsing step, the        wafers are dried, while still in the holder, using dry clean        nitrogen flow.    -   15. Post Exposure Bake: The wafers are then submitted to a 2        minute post exposure bake on a hotplate at 95 degree Celsius,        equally equipped with a vacuum mechanism.    -   16. Developing: The wafers still in the chemically resistant        holder are immersed in developer MF-321 (Shipley Chemicals,        Rohm-Haas) at room temperature for duration of 30 seconds.    -   17. Rinsing: The holder containing the wafers is removed from        the developer etchant bath and immerse in a bath of deionized        water. The wafers are left in this second bath for a 2 minutes        period with gentle manual agitation. The holder still containing        the wafers is removed from the bath, and sprayed with deionized        water to thoroughly finish the rinsing process.    -   18. Drying: Within a minute of the final rinsing step, the        wafers are dried, while still in the holder, using dry clean        nitrogen flow.    -   19. Surface Descum: The wafers are loaded in a Glenn 1000p        plasma asher run in ashing mode (Wafers on a grounded plate        below a powered plate), and submitted to a 30 seconds surface        descuming oxygen plasma at a pressure of 140 mTorr and a power        of 100 Watts forward power at 40 kHz. A flow of 18 sccm of        molecular oxygen is used.    -   20. Aluminium Evaporation: The wafers are loading in a metal        evaporator within 5 minutes of the surface descum process. A        layer of 100 nm of thermally evaporated Aluminium is now        deposited on the wafers. Evaporation is made at a pressure of no        less than 2 10ˆ-6 Torr at a rate of 25 Angstrom per seconds and        monitored using an Inficon controller.    -   21. Aluminium Thickness measurement: The thickness of the        aluminium is measured using a P-10 Profilometer (Tencor).    -   22. Zero Mode Waveguide Decasting: The Zero Mode Waveguide are        decasted from the enclosing Aluminium film by immersing them, in        a Teflon holder or other chemically resistant holder, in a bath        of 1165 Stripper (Shipley Chemicals, Rohm-Haas), or in a bath of        AZ-300T Stripper (Shipley Chemicals, Rohm-Haas). The bath is        submitted to sonication by immersing the Container holding both        the Stripper and the wafer holder in a sonicator. The wafers are        left in the decasting bath for 30 minutes or longer for about 45        minutes, and are provided with additional gentle agitation.    -   23. Rinsing: The stripping bath is removed from the sonicator.        The wafers are removed from the stripper bath and immerse in a        bath of deionized water. The wafers are left in this second bath        for a 2 minutes period with gentle manual agitation. The wafers        are removed from the bath, and sprayed with deionized water to        thoroughly finish the rinsing process.    -   24. Drying: Within a minute of the final rinsing step, the        wafers are dried, while still in the holder, using dry clean        nitrogen flow    -   25. Photoresist coating: The wafers are coated with Shipley 1827        photoresist spun at a speed of 1500 rpm. About 5 ml of resist is        dispensed. Acceleration and deceleration is set to 5 seconds.    -   26. Resist Bake: The wafers are baked on a CEE hotplate at a        temperature of 115 degree Celsius for 15 minutes. The plate is        equipped with a vacuum mechanism that allows good thermal        contact between the wafers and the hotplate surface.    -   27. Dicing: The wafer are diced using a K&S-7100 dicing saw        (Kulicke & Soffa) using a resin/diamond blade (ADT        00777-1030-010-QIP 600). The wafers are mounted on a low-tack        adhesive tape prior to dicing.    -   28. Die Removal: The dies are removed from the adhesive tape        manually and stored.    -   29. Resist removal: The layer of 1827 photoresist is removed by        immersing the dies first in an acetone bath for 1 minute, then        in a 2-propanol bath for 2 minute with gentle manual agitation.    -   30. Die Drying: The die is dried after being removed from the        2-propanol bath using dry clean air.    -   31. Plasma Clean: The wafers are loaded in a Drytek 100 plasma        etcher, and submitted to a 1 minute oxygen plasma at a pressure        of 140 mTorr, a molecular oxygen flow of 85 sccm oxygen and an        RF power of 500 Watts forward power at 13 Mhz. Alternatively,        Harrick Plasma Cleaner PDC-32G are submitted to a 5 minute dry        clean air plasma at a pressure of 2 Torr and 10.5 Watts of        power.

Example 2 Monitoring Enzymatic Synthesis of a DNA Strand by a Single DNAPolymerase Molecule in Real Time

This experiment can be performed using the optical system and reactionmixtures detailed below. However, the reference to any particularoptical system and parameter, buffer, reagent, concentration, pH,temperature, or the like, is not intended to be limiting. They areincluded to provide as one illustrative example of carrying out themethods of the present invention.

Enzymatic synthesis of a DNA strand by a single DNA polymerase moleculewas tracked in real time using a fluorescently labeled nucleotides.Individual Phi29^(N62D) DNA polymerase enzymes (Amersham Biosciences,Piscataway, N.J.) were immobilized in zero-mode waveguides (ZMWs) bynon-specific binding using a dilute enzyme solution. Afterimmobilization, the ZMW structures were washed to remove unbound enzyme,and then exposed to a solution containing the reaction reagents. As ofthe DNA template, a 70-bp pre-primed circular DNA sequence was used thatcontained two guanine bases in characteristic, asymmetric spacing (FIG.9A). Strand-displacement polymerizing enzymes such as Phi29 DNApolymerase will continuously loop around the circular template and thusgenerate a long and highly repetitive complementary DNA strand.

An R110-dCTP (Amersham Biosciences, Piscataway, N.J.) was used as thefluorescently-tagged nucleotide analog in which the fluorophore isattached to the nucleotide via a linker to the gamma-phosphate. Incontrast to the more commonly used base-labeled nucleotide analogs,gamma-phosphate-linked analogs are cleaved through the enzymaticactivity of DNA polymerase as the attached nucleotide is incorporatedinto the growing DNA strand and the label is then free to diffuse out ofthe effective observation volume surrounding the DNA polymerase. Theefficient removal of the fluorophore ensures continuously low backgroundlevels and prevents significant interference with DNA polymeraseactivity. These features of the gamma-phosphate-linked fluorophore arepreferable for this application because they will enable replacement ofall four bases with fluorophore-tagged analogs. Binding of a nucleotideand its subsequent incorporation into nucleic acid from a mismatch eventis distinguished because the rate constants of these two processes aresignificantly different, and because nucleotide incorporation involvesseveral successive steps that prevent zero delay time events.

All other nucleotides were supplied without labels. We have establisheda very effective way of removing any remaining trace amount of nativedNTP in a nucleotide analog preparation to ensure that errors are notintroduced due to the incorporation of unlabeled dNTPs by an enzymaticpurification using an alkaline phosphatase prior to the polymerizationassay.

To investigate the speed and processivity of the Phi29^(N62D) DNApolymerase under these conditions, the incorporation characteristicswere measured using R110-dCTP completely replacing dCTP in the reactionmixture, both in solution and with enzyme immobilized on a glasssurface. It was found that the enzyme efficiently utilized this analog,synthesizing complementary DNA of many thousands of base pairs in lengthwithout interruption in a rolling circle synthesis protocol, using bothsmall preformed replication forks (FIG. 9A) as well as larger circularDNA such as M13DNA. Only two, asymmetrically spaced R110-dCTPs were tobe incorporated into this template. Similar experiments demonstratedthat DNA polymerase can be immobilized to the bottom of ZMWs withoutlosing this catalytic activity.

The incorporation of the fluorescently labeled dCTP nucleotide wastracked during rolling-circle DNA synthesis by recording the fluorescentlight bursts emitted in an individual ZMW. DNA polymerase activity wasobserved in many waveguides as distinct bursts of fluorescence, whichlasted several minutes. The fluorescence time trace showed acharacteristic double burst pattern (FIG. 9B), each burst correspondingto an incorporation event of a R110-dCTP analog into the DNA strand andsubsequent cleavage of the fluorophore. In histograms of burst intervalsderived from the full time trace, two peaks corresponding to DNAsynthesis along the short (14 bases, approximately one second) and long(54 bases, approximately four seconds) DNA template segments arevisible, consistent with an overall average speed measured in bulksolution under these conditions of approximately ten base pairs persecond.

It is noteworthy that this single-molecule activity at a fluorophoreconcentration of 10 μM was readily observable. In conventionally createdexcitation volumes, the number of fluorophores would be far too high topermit the observation of individual enzymatic turnovers of DNApolymerase. These experiments thus confirmed the validity of theZMW-based single-molecule DNA sequencing approach by verifying that (a)immobilization of DNA polymerase in ZMWs does not affect its enzymaticactivity; (b) fluorescent gamma-phosphate-linked nucleotide analogs donot inhibit the activity of DNA polymerase; and (c) ZMWs provide anadequate degree of confinement to detect single-molecule DNA polymeraseactivity at physiological concentrations of reagents. More generally,these results prove that ZMWs allow single-molecule analysis of enzymekinetics, especially involving any enzyme that can be attached to thesurface and for which substrates can be fluorescently labeled.

Example 3 Real Time Sequencing Using Multiple Different LabeledNucleotides

An experiment similar to that described in Example 2, above, wasperformed using two different labeled nucleotide analogs. The experimentcan be performed using the optical setup or system and reaction mixturesdetailed as follows. However, the reference to any particular opticalsetup and parameter, buffer, reagent, concentration, pH, temperature, orthe like, is not intended to be limiting. They are included to provideas one illustrative example of carrying out the methods of the presentinvention.

Preparing reaction samples: Approximately 10 μl of reaction mixture isused in one sequencing reaction the reaction mixture generally contains0.5-1 mM MnCl₂, 0.1-1 uM DNA template, 10 μM dATP, 10 μM dGTP, 10 μMSAP-treated Alexa 488-dC4P, and 10 μM SAP-treated Alexa 568-dT4P, andDNA polymerase. The labeled dC4P and dT4P can also be substituted withlabeled dA4P and dG4P.

Preparing Zero-mode waveguide: Prior to the polymerization reaction, azero-mode waveguide is typically refreshed in a plasma cleaner. A PDMSgasket covering the ZMW is placed onto the waveguide to cover theindividual optical confinements. An aliquot of the reaction mixturedescribed above except the DNA polymerase is applied without touchingthe waveguide surface. The diffusion background is measured. If thebackground (i.e., fluorescence burst from the ZMW) low and acceptable,then DNA polymerase will be applied to ZMW and immobilized thereon. Theimmobilization mixture typically contains 0.5 to 1 mM MnCl₂, 0.1 to 1 uMtemplate, 15 nM DNA polymerase, in a buffer of 25 mM Tris-HCL, pH 7.5and 10 mM beta-mercaptoethanol. The polymerase is allowed to stick tothe surface of ZMW after an incubation of about 15 minutes at about 0°C. The immobilization reaction mixture is then removed, replaced withthe reaction mixture described above.

A microscope system equipped with an appropriate laser, e.g., Ar/Krlaser, is used that includes an optical setup for simultaneouscollection and detection of signals from multiple different waveguides,and for the resolution of each of the A488 and 568 fluorophores present.The system includes an objective lens and a series ofdichroics/notch-off filters for separating emitted fluorescence fromreflected excitation light. The emitted signals are passed through awedge filter to spatially separate the signal component of eachfluorophore, and each signal is imaged onto an EMCCD camera.

Polymerase Activity Measurement: The ZMW is placed under the microscope.Polymerization reaction is then monitored using a camera for a desiredperiod of time, e.g., two minutes or longer, after the transilluminationlight is applied. The data is automatically transmitted to a computerthat stores and trace the fluorescence burst of each reaction in theZMW.

A circular DNA having either a block of repeating A bases followed by ablock of G bases, or a series of repeating A-G bases was sequencedaccording to the aforementioned procedures. A representative profile ofthe fluorescence bursts corresponding to each incorporation event of thelabeled nucleotides is depicted in FIG. 16, which indicates thatreal-time and single-molecule sequencing has been achieved with morethan one type of labeled nucleotides. Statistical analysis of pulse datafrom multiple separate repeats and multiple different waveguidesestablishes the sequence dependant detection of incorporation of labeledbases in real time.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. To the extent not already expressly incorporatedherein, all published references and patent documents referred to inthis disclosure are incorporated herein by reference in their entiretyfor all purposes.

1. An apparatus comprising: an array of optical confinements having adensity on a substrate exceeding 4×10⁴ confinements per mm², whereinsaid optical confinements are suitable for holding a biological reagent,and wherein said optical confinements provide an effective observationvolume that permits observation of individual molecules present in saidbiological reagent; and an optical system operatively coupled to theoptical confinements that detects signals from the effective observationvolume of said confinements.
 2. The apparatus of claim 1, wherein saiddensity exceeds 10⁵ confinements per mm².
 3. The apparatus of claim 1 or2, wherein said optical confinements hold a biological reagent.
 4. Theapparatus of claim 3, wherein said biological reagent comprises anenzyme.
 5. The apparatus of claim 3, wherein said biological reagentfurther comprises substrates of said enzyme present at a concentrationof about Michaelis constant (Km) of said enzyme for said substrates. 6.The apparatus of claim 5, wherein said concentration is higher thanabout 1 micromolar.
 7. The apparatus of claim 5, wherein saidconcentration is higher than about 50 micromolar.
 8. The apparatus ofclaim 5, wherein said substrates are labeled.
 9. The apparatus of claim5, wherein said individual molecules are products of a reaction betweensaid enzyme and said substrates contained in said biological reagent.10. The apparatus of claim 3, wherein said individual molecules areDNA/polymerase complexes.
 11. The apparatus of claim 3, wherein saidindividual molecules are biomolecules selected from the group consistingof nucleic acids, polypeptides, and combinations thereof.
 12. Theapparatus of claim 3, wherein said individual molecules are organic orinorganic chemical compounds.
 13. The apparatus of claim 3, wherein atleast one individual confinement is separated from at least one otheroptical confinement in said array by a distance less than about 1000 nm.14. The apparatus of claim 3, wherein said optical confinements in saidarray are separated from each other by a distance between about 200 nmand about 1000 nm.
 15. The apparatus of claim 3, wherein said arraycomprises at least about 2×10⁵ optical confinements.
 16. The apparatusof claim 3, wherein said array comprises at least about 10⁷ opticalconfinements.
 17. The apparatus of claim 3, wherein said individualmolecules are labeled with a detectable label.
 18. The apparatus ofclaim 17, wherein said detectable label is selected from the groupconsisting of a fluorescent label, a chemiluminescent label, and aradioactive label.
 19. The apparatus of claim 3, wherein said opticalconfinements are made of porous film.
 20. The apparatus of claim 3,wherein each individual confinement in said array is operatively coupledto an optical transmission element transmitting an incident light. 21.The apparatus of claim 3, wherein said optical system further comprisesa photon detector.
 22. The apparatus of claim 3, wherein said opticalconfinements are waveguides comprising a cladding surrounding a core,wherein said cladding precludes propagation of a majority of incidentlight having a wavelength greater than a cutoff wavelength through saidcore.
 23. The apparatus of claim 22, wherein said waveguides precludepropagation of more than 90% of incident light having a wavelengthgreater than a cutoff wavelength through said core.
 24. The apparatus ofclaim 22, wherein said waveguides preclude propagation of more than 99%of incident light having a wavelength greater than a cutoff wavelengththrough said core.
 25. The apparatus of claim 22, wherein said claddingcomprises a sheet, and one or more holes extending through said sheetwhere each hole constitutes the core.
 26. The apparatus of claim 22,wherein said core is cylindrical in shape.
 27. The apparatus of claim22, wherein said core is non-cylindrical in shape.
 28. The apparatus ofclaim 22, wherein said cladding comprises a metal alloy.
 29. Theapparatus of claim 28, wherein said alloy is aluminum alloy.
 30. Theapparatus of claim 3, wherein each individual confinement provides aneffective observation volume less than about 1000 zeptoliters.
 31. Theapparatus of claim 3, wherein each individual confinement provides aneffective observation volume less than about 80 zeptoliters.
 32. Theapparatus of claim 3, wherein each individual confinement provides aneffective observation volume less than about 10 zeptoliters.
 33. A kitcomprising an array of optical confinements of claim 3 and aninstruction manual for use of said array.
 34. The apparatus of claim 3,wherein a plurality of said optical confinements in said array eachholds a single complex of a target nucleic acid and a polymerizationenzyme, and more than one type of labeled nucleotides or nucleotideanalogs present at a concentration about 1 uM to about 50 uM.
 35. Theapparatus of claim 34, wherein said optical confinements are zero-modewaveguides.
 36. A method of detecting a biological analyte, comprising:optically capturing the analyte within an optical confinement that iscreated by (a) providing an array of optical confinements having adensity on a substrate exceeding 4×10⁴ confinements per mm², whereinsaid optical confinements provide an effective observation volume thatpermits observation of individual molecules; and an optical systemoperatively coupled to the optical confinements that detects signalsfrom the effective observation volume of said confinements; and (b)illuminating at least one optical confinement within the array that issuspected to contain the analyte with an incident light beam therebydetecting the analyte.
 37. The method of claim 36, wherein said densityon a substrate exceeding 10⁵ confinements per mm².
 38. The method ofclaim 36, wherein said analyte is labeled with a detectable label. 39.The method of claim 36, wherein said analyte is selected from the groupconsisting of nucleic acid, polypeptide, organic compound, and inorganiccompound.
 40. A method of performing multiple chemical reactionsinvolving a plurality of reaction samples, comprising: (a) providing anarray of optical confinements of claim 1; (b) placing the plurality ofreaction samples comprising labeled reactants into the opticalconfinements in the array, wherein a separate reaction sample is placedinto a different confinement in the array; (c) subjecting the array toconditions suitable for formation of products of the chemical reactions;and (d) detecting the formation of the products with said opticalsystem.
 41. The method of claim 40, wherein the step of detectingcomprises illuminating the different confinements with an incident lightbeam and detecting an optical signal emitted from the reaction samples.42. The method of claim 40, wherein the chemical reactions involveprotein-protein interactions.
 43. The method of claim 40, wherein thechemical reactions involve nucleic acid-protein interactions.
 44. Themethod of claim 40, wherein the chemical reactions involve nucleicacid-nucleic acid interactions.
 45. A method of sequencing a pluralityof target nucleic acid molecules, comprising: (a) providing an array ofoptical confinements having a density on a substrate exceeding 4×10⁴confinements per mm², wherein said optical confinements provide aneffective observation volume that permits observation of individualmolecules; and an optical system operatively coupled to the opticalconfinements that detects signals from the effective observation volumeof said confinement; (b) mixing in the optical confinements theplurality of target nucleic acid molecules, primers complementary to thetarget nucleic acid molecules, polymerization enzymes, and more than onetype of nucleotides or nucleotide analogs to be incorporated into aplurality of nascent nucleotide strands, each strand being complementaryto a respective target nucleic acid molecule; (c) subjecting the mixtureof step (b) to a polymerization reaction under conditions suitable forformation of the nascent nucleotide strands by template-directedpolymerization of the nucleotides or nucleotide analogs; (d)illuminating the optical confinements with an incident light beam; and(e) identifying the nucleotides or the nucleotide analogs incorporatedinto the each nascent nucleotide strand.
 46. The method of claim 45,wherein the step of identifying yields a time sequence of incorporationof the nucleotides provided in step (b).
 47. The method of claim 45,wherein the identifying step is effected while the template-directedpolymerization is taking place.
 48. The method of claim 45, wherein thetarget nucleic acid molecules are selected from the group consisting ofcircular DNA molecules, linear DNA molecules, RNA molecules, and DNA/RNAhybrids.